Methods of treatment by targeting vcam1 and maea

ABSTRACT

Antibodies and antibody fragments that inhibit the activity of vascular cell adhesion molecule 1 (VCAM1) and/or macrophage erythroblast attacher (MAEA) are provided, along with formulations and kits comprising these antibodies and antibody fragments and the use of the disclosed compositions, formulations, and kits to treat cancers, sickle cell disease, and Polycythemia Vera.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.16/773,907, filed Jan. 27, 2020, which is a continuation-in-part of U.S.patent application Ser. No. 16/199,555, filed Nov. 26, 2018, nowabandoned, which is a continuation-in-part of and claims priority of PCTInternational Patent Application No. PCT/US2017/034365, filed May 25,2017, which designates the United States of America and which claims thebenefit of U.S. Provisional Patent Application No. 62/342,360, filed onMay 27, 2016, the contents of which are incorporated herein byreference.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant numbersHL116340, HL069438 and DK056638 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

STATEMENT OF SEQUENCE LISTING

This application contains an ST.26 compliant Sequence Listing, which wassubmitted in xml format via Patent Center and is hereby incorporated byreference in its entirety. The .xml copy, created on Dec. 27, 2022 isnamed Sequence_Listing.xml and is 131 KB in size.

BACKGROUND

Throughout this application various publications are referred to insuperscripts. Full citations for these references may be found at theend of the specification before the claims. The disclosures of thesepublications are hereby incorporated by reference in their entiretiesinto the subject application to more fully describe the art to which thesubject application pertains.

Hematopoietic stem cells (HSCs) possess the ability to maintain theentire population of blood cells throughout life and to replenish thehematopoietic system after transplantation into marrow-ablatedrecipients. During fetal and adult life, HSCs are able to migrate toectopic niches via the blood stream. Once in the blood, HSCs home toperivascular stromal and endothelial cells expressing adhesionmolecules, then navigate the vascular networks of the marrow, spleen,and liver before returning to potential bone marrow niches.

Under homeostasis, HSCs reside in the specialized bone marrow (BM) nichecomposed of various cellular and molecular constituents. Whereasmesenchymal stem and progenitor cells provide most niche factor activitycontributing to HSC maintenance, differentiated hematopoietic cells suchas macrophages can regulate indirectly HSC retention in BM via theniche. In addition, macrophages tightly interact with red blood cellprecursors to form a structure known as the erythroblastic island (EI)in which interactions via vascular cell adhesion molecule 1 (VCAM1)and/or macrophage-erythroblast attacher (MAEA, also called EMP) arethought to play important roles in the terminal maturation oferythroblasts. The attachment of the developing erythroblasts (EBs) tothe central macrophages within the islands is critical for survival,proliferation, and proper differentiation of developing erythrocytesboth in vitro and in vivo.

VCAM1 is an adhesion molecule expressed by BM stromal and endothelialcells and certain classes of hematopoietic cells. VCAM1's major ligandis integrin α4β1 (also known as Very Late Antigen-4, “VLA-4”). Theinteraction between VCAM1 and VLA-4 mediates cell-cell interaction inmultiple cell types, and both VCAM1 and VLA-4 have been implicated inHSC homing and retention into the bone marrow and mobilization into theperipheral blood.

VCAM1 protein mediates the adhesion of lymphocytes, monocytes,eosinophils, and basophils to the vascular endothelium. VCAM1 alsofunctions in leukocyte-endothelial cell signal transduction, and it mayplay a role in the development of atherosclerosis and rheumatoidarthritis (RA).

MAEA is an adhesion molecule originally identified on macrophages anderythroblasts, and it is suggested to play a role in the formation ofEIs. However, its function in the adult hematopoietic system is unknowndue to the perinatal death of MAEA-deficient mice. Other candidatemolecules, e.g., VCAM1, have also been suggested to participate in EIformation, but cell type-specific requirement of these molecules for EIformation and function in vivo has not been examined.

SCD is a blood disorder that causes red blood cells (RBCs) to have anabnormal “sickled” shape that is rigid.^(121,124,125) Whereas RBCs inhealthy individuals are elastic, sickled RBCs are rigid, and studiessuggest that this loss of elasticity of RBCs is central to SCD. SCD isassociated with a number of chronic and acute symptoms.¹²⁶

The present disclosure provides anti-VCAM1 and anti-MAEA antibodies,formulations and kits comprising anti-VCAM1 and anti-MAEA antibodies,and therapies for treatment of hematological malignancies and othercancers as well as anti-VCAM1 therapies for treatment of sickle celldisease (SCD).

SUMMARY

The present disclosure provides methods of treating condition in asubject comprising administering to the subject an antibody or antibodyfragment in an amount effective to inhibit the activity of vascular celladhesion molecule 1 (VCAM1) and/or an antibody or antibody fragment inan amount effective to inhibit the activity of macrophage erythroblastattacher (MAEA) to treat a condition in a subject, wherein the antibodyor antibody fragment is specific for VCAM1 or MAEA. For example,provided herein are methods of treating a condition in a subject,wherein the condition is a cancer (e.g., a hematologic malignancy ormyeloproliferative disease). Also provided herein are methods fortreating a condition, wherein the condition is sickle cell disease(SCD).

Also provided are methods of inhibiting engraftment of leukemia cells(e.g., acute myeloid leukemia (AML), chronic myeloid leukemia (CML),acute lymphoblastic leukemia (ALL), or chronic lymphocytic leukemia(CLL) cells) in a subject, the methods comprising administering to thesubject an antibody or antibody fragment in an amount effective toinhibit the activity of VCAM1 and/or an antibody or antibody fragment inan amount effective to inhibit the activity of MAEA to inhibit leukemia(e.g., AML, CML, ALL or CLL) cell engraftment in a subject, wherein theantibody or antibody fragment is specific for VCAM1 or MAEA.

Still further provided are methods of enhancing the efficacy ofcytarabine for treating a cancer in a subject, comprising administeringto the subject an antibody or antibody fragment in an amount effectiveto inhibit the activity of VCAM1 and/or an antibody or antibody fragmentin an amount effective to inhibit the activity of MAEA in combinationwith cytarabine to enhance the efficacy of cytarabine for treating acancer in a subject. wherein the antibody or antibody fragment isspecific for VCAM1 or MAEA.

Sickle cell disease (SCD), caused by a single missense mutation in theβ-globin gene, affects around 100,000 patients in the United States andmillions worldwide.⁴⁶⁻⁴⁷ Mutated β-globin polymerizes underdeoxygenation, leading to sickle-shaped RBCs, which exhibit increasedadherence to other blood cells or to the endothelium, and are prone toundergo premature clearance and hemolysis.⁴⁸ The RBC alterations lead toa chronic inflammatory state resulting in ischemic tissue damagemanifested by severe pain and organ failures. The pathophysiology of VOEand chronic organ damage is complex and involves the interplay ofaltered blood rheology, endothelial activation, and the secretion ofinflammatory cytokines enabling leukocyte adhesion and activation.⁴⁹Intravital microscopy analysis of the SCD mouse microcirculation hasrevealed that RBCs interact with adherent leukocytes in the inflamedvasculature.⁵⁰ The accumulation of activated neutrophils interactingwith RBCs progressively reduces blood flow and produces venularocclusions.^(51,52)

Polycythemia Vera (PV) is a myeloproliferative neoplasm (MPN)characterized by elevated erythropoiesis associated with aconstitutively active mutant form of JAK2 tyrosine kinase, JAK2^(V617F)PV patients show splenomegaly, expansion of erythroid progenitors andincrease in reticulocytosis, of which only some can be managed byphlebotomy, hydroxyurea or JAK2 inhibitors.^(53,54,55,56)

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C. FIGS. 1A-1C depict experimental results demonstrating thatVCAM1 is expressed at higher levels on acute myelogenous leukaemia (AML)stem cells than on healthy hematopoietic stem cells. FIG. 1A is a chartdepicting the percentage of VCAM1⁺ cells within hematopoietic stem cells(HSC) and multipotent progenitors (MPP) from the bone marrow (BM),spleen, and blood (n=6-18). FIG. 1B depicts a schematic overview ofexperimental strategy to generate mouse leukemic MLL-AF9 cells. FIG. 1Cis a chart depicting the median fluorescence intensity (MFI) of VCAM1 onbulk control healthy and leukemic MLL-AF9 BM cells (left panel), andhealthy HSCs and leukaemia stem cells (LSCs, right panel). LSCs werephenotypically defined as Lineage⁻ IL7Rα⁻ Sca1⁻ MLL-AF9 GFP⁺c-Kit^(high) CD34^(low) FcγRII/III^(high) cells. (n=6-9); MPP (LSKCD150⁻ CD48⁻); HSC (LSK CD150⁺ CD48⁻).

FIGS. 2A-2G. FIGS. 2A-2G depict experimental results demonstrating thatVCAM1 endogenous deletion does not cause significant hematopoieticdefects. FIG. 2A is a chart depicting fluorescent-activated cell sorting(FACS) analysis of the BM of Csf1r-iCre;loxp-TdTomato transgenic miceshowing the recombination efficiency of Csf1r-iCre in phagocytes, HSCand MPP (n=3-6). FIG. 2B depicts experimental results demonstrating thatVCAM1 is efficiently depleted in VCAM1^(Δ/Δ) BM HSCs, as seen by FACS(n=4-13) and mRNA (n=4-6) analyses (absolute number of BMNCs, HSCs andMPPs per femur in control and VCAM1^(Δ/Δ) mice (n=5-6)). FIG. 2D is achart depicting experimental results demonstrating that colony output onday 7 of BM colony-forming unit in culture from control and VCAM1^(Δ/Δ)mice (n=3), (GEMM: granulocyte, macrophage, erythroid and megakaryocyte;GM: granulocyte and macrophage; M: macrophage; G: granulocyte; BFU-E:erythroid). FIG. 2E is a chart depicting experimental results comparingconcentration of white blood cells (WBC), erythrocytes (RBC) andplatelets (PLTs) in the blood of VCAM1^(Δ/Δ) mice as compared tolittermate controls (n=12). FIG. 2F Concentration of HSCs and MPPs inthe blood of VCAM1^(Δ/Δ) mice as compared to littermate VCAM1 floxedcontrols (n=3). FIG. 2G depicts a pair of charts quantifying spleencellularity (left) and absolute number of HSC and MPP (right) per spleenin control and VCAM1^(Δ/Δ) mice (n=5). Error bars, mean±s.e.m. *p<0.05,**p<0.01, ****p<0.0001; unpaired Student's t test.

FIGS. 3A-3E. FIGS. 3A-3E depict experimental results showing thatVCAM1-deficient HSCs exhibit normal viability, cell cycle, andproliferation. FIG. 3A depicts a chart showing the percentage of viable(Annexin V⁻ DAPI⁻) HSC and MPP in the BM of control and VCAM1^(Δ/Δ) mice(n=3). FIG. 3B is a chart showing cell cycle analysis, using anti-Ki67and Hoechst 33342 staining of HSCs from control and VCAM1^(Δ/Δ) mice(n=3-4). FIG. 3C is a chart showing the percentage of proliferating HSCin the BM of control and VCAM1^(Δ/Δ) mice, as determined by BrdUincorporation (n=4). FIG. 3D Quantitative PCR (qPCR) analysis of cellcycle regulator genes within sorted HSCs from control and VCAM1^(Δ/Δ)mice. FIG. 3E depicts a series of charts showing the number of BMNCs,MPP and HSC per femur in control and VCAM1^(Δ/Δ) mice after 5-FUinjection (n=3-5). Error bars, mean±s.e.m. Non-significant (ns);*p<0.05. Unpaired Student's t test (FIGS. 3A-3E).

FIGS. 4A-4E. FIGS. 4A-4E depict experimental results illustrating thatblocking anti-VCAM1 antibody treatment decreases the number of leukaemiastem cells and synergizes with cytarabine in vivo. FIG. 4A is agraphical depiction of an outline of experiment strategy. Moribund sicksecondary recipient leukemic mice were daily injected with IgG control(100 μg), anti-VCAM1 antibody (100 μg), cytarabine (100 mg/kg) or acombination of anti-VCAM1/cytarabine for 5 days. Mice were analysed byFACS 1 day after the last injection. FIG. 4B is a series of chartsshowing BM cellularity, absolute number, and percentage of bulkMLL-AF9-GFP⁺ cells and LSCs in the BM of control and treatment groups(n=5-6). FIG. 4C is a chart showing the percentage of MLL-AF9-GFP⁺ cellsin the blood of recipient hosts comparing pre- and post-treatment (n=5).FIG. 4D depicts survival curves of leukemic treatment groups. Arrowpoints to the beginning of treatment. IgG and anti-VCAM1 wereadministered during 10 consecutive days every and cytarabine groupsduring 5 consecutive days. All treatments were repeated after 4 weeks.FIG. 4E depicts a series of charts quantifying BM cellularity (left) andabsolute number of HSC, MPP, and LSK per femur (middle) and per mL ofblood (right) in healthy C57BL/6 mice treated for 5 days with dailyinjections of either anti-VCAM1 or IgG control antibody (100 μg) (n=5).Error bars, mean±s.e.m. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001;unpaired Student's t test (FIG. 4E) and paired Student's t test (FIG.4C). One-way ANOVA analyses followed by Tukey's multiple comparisontests were in (FIG. 4B). Log-rank analysis was used for the Kaplan-Meiersurvival curves in (FIG. 4D).

FIGS. 5A-5E. FIGS. 5A-5E are a series of figures showing that treatmentof healthy wild-type mice with a blocking anti-VCAM1 monoclonalantibody. FIG. 5A depicts an outline of experiment strategy. FIG. 5Bdepicts experimental results from BM cells from treated groups that wereincubated with an anti-rat antibody and after washing stained forphenotypic HSCs and probed for VCAM1 expression. FIG. 5C depicts a chartquantifying cells per mL of blood in healthy C57BL/6 mice treated for 5days with daily injections of either anti-VCAM1 or IgG control antibody(n=5). FIG. 5D is a chart depicting body, liver, and spleen weight ofIgG and anti-VCAM1-treated mice from FIG. 5A. FIG. 5E is a tabledepicting hematology lab analysis results of peripheral blood drawnpost-treatment. White blood cell (WBC), red blood cell (RBC), hemoglobin(HGB), hematocrit (HCT), platelets (PLT), neutrophils (Neut.),lymphocytes (Lymph.), reticulocytes (Retic.). P-values of IgG comparedto anti-VCAM1 treated mice are shown for each parameter (n=5). Errorbars, mean±s.e.m. Non-significant (ns); unpaired Student's t test.

FIGS. 6A-6D. FIGS. 6A-6D are a series of figures showing that high VCAM1expression is associated with poor prognosis in human AML patients.FIGS. 6A and 6B are charts depicting experimental results ofKaplan-Meier (FIG. 6A) overall and (FIG. 6 -B) disease free survival ofAML patients (TCGA, Ley et al., 2013) with high and low VCAM1 expression(mRNA expression z-Score threshold±2). FIGS. 6C and 6D depict survivalcurves of NSG mice transplanted with primary human AML samples andtreated with (FIG. 6C) control IgG or anti-VCAM1 antibody or (FIG. 6D)with cytarabine or the combination cytarabine/anti-VCAM1. Log-rankanalysis was used for the Kaplan-Meier survival curves to calculate pvalue.

FIGS. 7A, 7B. FIGS. 7A and 7B depict VCAM1 expression in human cancercell lines. FIG. 7A depicts a pie chart showing VCAM1 expression statusacross 675 human cancer cell lines. FIG. 7B is a chart showing meandistribution of VCAM1 expression (reads per kilobase of transcript permillion mapped reads—RPKM) per human cancer cell line, grouped bymetastatic tissue of origin.⁴⁵

FIG. 8 . FIG. 8 depicts a chart showing VCAM1 genetic alterations inprimary human cancer tissues. The chart displays a cross-canceralteration summary for VCAM1 from 126 human cancer genomics studiesgenerated by cBioPortal for Cancer Genomics from MSKCC.

FIGS. 9A-9F. FIGS. 9A-9G depict experimental result showing that VCAM1provides “don't-eat-me” recognition. FIG. 9A depicts charts showing theconcentration of HSCs, MPPs and colony-forming unit cell (CFU-C) in theblood of VCAM1^(Csf1r-iCre) mice as compared to littermate VCAM1^(fl/fl)(n=3-4). FIG. 9B depicts a chart showing the time course of bloodchimerism post-transplant of VCAM1^(fl/fl) and VCAM1^(Csf1r-iCre) donorcells. FIG. 9C depicts a series of charts showing in vivo phagocytosisassay comparing VCAM1^(fl/fl) and VCAM1^(Csf1r-iCre) Lineage⁻ cellslabelled with CFDA-SE and transplanted. Recipient mice were sacrificed 4days after and the percentage of recipient CD45.1 CFDA-SE⁺ phagocyticcells determined by FACS in the BM and spleen (n=4-5). FIG. 9D depicts arepresentative immunofluorescence image of splenic F4/80⁺ macrophageswith VCAM1^(Csf1r-iCre) phagocytosed CFDA-SE⁺ cells (arrowheads). FIG.9E depicts a chart showing experimental results where blood circulationwas connected between CD45.2 VCAM1^(fl/fl) and VCAM1^(Csf1r-iCre) miceand CD45.1 mice by parabiotic surgery and mobilization induced by G-CSFinjection. Frequency of donor CD45.2 HSC and MPP from VCAM1^(fl/fl) andVCAM1^(Csf1r-iCre) that homed and engrafted in the BM of CD45.1 pairedmice, one week after the last G-CSF injection (n=3). FIG. 9F depicts achart showing examples of BM HSC chimerism plots for VCAM1^(fl/fl) andVCAM1^(Csf1r-iCre) donors. MPP (LSK CD150⁻ CD48⁻); HSC (LSK CD150⁺CD48⁻). Error bars, mean s.e.m. Unpaired Student's t test (FIGS. 9A-9C,9E).

FIGS. 10A-10H. FIGS. 10A-10H depict experimental results showing thatVCAM1 is essential for HSC engraftment in haplotype-mismatchedtransplantation. FIG. 10A depicts a schematic diagram and survival curveshowing VCAM1^(fl/fl) and VCAM1^(Csf1r-iCre) syngeneic (H-2^(b/b)) andhaplotype-mismatched (H-2^(b/q)) lines used. H-2^(b) (C57BL/6 strain)and H-2^(q) (FVB/N strain). Survival curve of recipient mice givenlethal radiation and transplanted with 2 million BMNCs, non-competitivetransplantation (n=6-11). FIGS. 10B and 10C depict charts showingquantification of long-term haematopoietic reconstitution from syngeneicand haplotype-mismatched VCAM1^(fl/fl) and VCAM1^(Csf1r-iCre) cells bycompetitive reconstitution assays in the blood (FIG. 10B) and in the BM(FIG. 10C), 16 weeks post-transplantation (n=6-14). FIG. 10D depicts achart showing quantification of tri-lineage (My, myeloid; B cell and Tcell) engraftment in the mice analyzed in FIGS. 10B and 10C. FIG. 10Edepicts a chart showing percentage of BM immune cells double positivefor VLA4 and PIR-B, as determined by FACS (n=3). FIG. 10F depicts aseries of charts showing phospho-flow analysis of tyrosinephosphorylation (P-TYR) levels in host CD45.1⁺ PIR-B⁺ phagocytic cellstransplanted with haplotype-mismatched CD45.2 VCAM1^(fl/fl) andVCAM1^(Csf1r-iCre) cells. P-TYR levels are represented as medianfluorescent intensity (MFI) normalized to the basal P-TYR levels ofphagocytic cells in Pirb^(−/−) mice (n=5). FIG. 10G depicts arepresentative FACS histogram for P-TYR levels in host CD45.1⁺ Gr1highPIR-B⁺ monocytes from the mice in FIG. 10F. FIG. 10H depicts a chartshowing quantification of hematopoietic reconstitution from syngeneicand haplotype-mismatched VCAM1^(fl/fl) and VCAM1^(Csf1r-iCre) cells bycompetitive reconstitution assays into Pirb^(−/−) mice, 1 weekpost-transplantation (n=6). Donor cell engraftment was evaluated bydetecting the levels of donor male DNA in the female recipient blood, byreal-time PCR. Error bars, mean±s.e.m. unpaired Student's t test (FIGS.10B, 10F). One-way ANOVA analyses followed by Tukey's multiplecomparison tests (FIGS. 10C, 10D, 10H). Log-rank analysis was used forthe Kaplan-Meier survival curves in (FIG. 10A).

FIGS. 11A-11F. FIGS. 11A-11F depict experimental results showing thatloss of VCAM1 inhibits the establishment and progression ofMLL-AF9-induced AML and markedly improved survival in mouse model. FIG.11A depicts charts showing analysis of AML primary recipientstransplanted with haplotype-mismatched VCAM1^(fl/fl) andVCAM1^(Csf1r-iCre) transduced MLL-AF9-GFP LSKs, after 55 days. BMcellularity and percentage of AML-GFP⁺ cells and LSCs (n=5). FIG. 11Bdepicts representative images of a sternal BM segment from the miceanalyzed in FIG. 11A. FIG. 11C depicts experimental results frompre-leukemic haplotype-mismatched VCAM1^(fl/fl) and VCAM1^(Csf1r-iCre)luciferase expressing AML cells that were transplanted into sub-lethallyirradiated mice and leukemia progression was quantified bybioluminescence (n=5). Luciferase imaging of representative mice fromeach group is shown at week 10 post-transplant. FIG. 11D depicts a chartshowing Kaplan-Meier survival analysis of secondary recipient micereceiving 20,000 GFP⁺ leukemia cells from haplotype-mismatchedVCAM1^(fl/fl) and VCAM1^(Csf1r-iCre) primary recipients. FIG. 11Edepicts charts showing the BM cellularity and absolute number ofmacrophages per femur in C57BL/6 mice treated with PBS or clodronateliposomes and transplanted with VCAM1^(fl/fl) and VCAM1^(Csf1r-iCre)pre-leukemic cells, at week 4 (n=5). FIG. 11F depicts a chart showingthe percentage of haplotype-mismatched AML-VCAM1^(fl/fl) orAML-VCAM1^(Csf1r-iCre) GFP⁺ cells in the blood of recipient hoststreated with weekly injections of PBS or clodronate liposomes (n=5-10).Error bars, mean±s.e.m. unpaired Student's t test (FIGS. 11A, 11C, 11F).Log-rank analysis was used for the Kaplan-Meier survival curves in FIG.11D. One-way ANOVA analyses followed by Tukey's multiple comparisontests were used in FIG. 11E.

FIGS. 12A-12C. FIGS. 12A-12C depict experimental results showing thatanti-VCAM1 antibody treatment blocks human primary AML progression andextends the survival of NSG-transplanted mice. FIG. 12A depicts aschematic outline of experimental strategy. NSG mice were transplantedwith primary human AML and upon disease establishment were dailyinjected with IgG1 control (100 μg) or anti-human VCAM1 antibody (100μg) for 10 days. FIG. 12B depicts a chart showing survival of leukemicmice treated in FIG. 12A. FIG. 12C depicts a schematic diagram showingimmune cells and receptors mediating the cooperative anti-phagocyticactivity of VCAM1 and MHC-I enabling “don't-eat-me” or “kill-me”activity. Log-rank analysis was used for the Kaplan-Meier survivalcurves (FIG. 12B).

FIGS. 13A-13D. FIGS. 13A-13D depict experimental results showing VCAM1is expressed on HSCs and progenitor cells. FIGS. 13A and 13B depictgating strategies for the analyses of HSC and progenitor populations.FIG. 13C depicts representative histograms of VCAM1 expression levels inthe populations represented. FIG. 13D depicts a chart showing thepercentage of VCAM1 positive cells within progenitor cell populationsfrom the bone marrow (BM) and spleen (n=3).

FIGS. 14A-14D. FIGS. 14A-14D depict experimental results demonstratingthat VCAM1-deficient HSCs exhibit normal viability, cell cycle, andproliferation. FIG. 14A depicts a schematic overview of experimentalstrategy. FIG. 14B depicts a survival curve of recipient mice givenlethal radiation and transplanted with 2 million BM nuclear cells(BMNCs) from VCAM1^(fl/fl) (control) and VCAM1^(Csf1r-iCre) mice,non-competitive transplantation (n=7-8). FIG. 14C depicts a schematicoverview of experimental strategy. FIG. 14D depicts a chart showingquantification of long-term reconstituting HSCs from VCAM1^(fl/fl) andVCAM1^(Csf1r-iCre) mice by competitive reconstitution assay in the blood(n=9-15).

FIGS. 15A-15C. FIGS. 15A-15C depict experimental results demonstratingthat the distribution of HSCs in the mouse BM is not altered after VCAM1deletion in Csf1r-iCre⁺ cells. FIG. 15A depicts representativewhole-mount images of the sternal BM of control and VCAM1^(Csf1r-iCre)mice and magnified high power view. The dashed outline denotes bone-BMborder. Arterioles (Art) are identified by CD31⁺ CD144⁺ Sca1⁺expression. Phenotypic HSC are identified by Lineage-CD41⁻ CD48⁻ CD150⁺expression, and megakaryocytes (Mk) are distinguished by their size,morphology, and CD41⁺ CD150⁺ expression. FIGS. 15B and 15C depict chartsquantifying the localization of HSCs relative to arterioles (FIG. 15B)and Mks (FIG. 15C) in VCAM1^(fl/fl) and VCAM1^(Csf1r-iCre) mice. Errorbars, mean±s.e.m. Two-sample Kolmogorov-Smirnov tests were used forcomparisons of distribution patterns in FIGS. 15B and 15C.

FIGS. 16A-16C. FIGS. 16A-16C depict experimental results showing thatVCAM1 acts as a “don't-eat-me” signal. FIG. 16A depicts a chartquantifying the absolute number of donor (CD45.2) VCAM1^(fl/fl) andVCAM1^(Csf1r-iCre) cells that homed to the BM of lethally irradiatedCD45.1 recipients, 3 hours after injection (n=5). FIG. 16B depictsrepresentative FACS plots of the in vivo phagocytosis assay in FIG. 9C.FIG. 16C depicts a schematic outline of experiment strategy. Lethallyirradiated CD45.1 recipients were transplanted competitively with 2million of VCAM1^(Csf1r-iCre) or VCAM1^(fl/fl) BMNCs. At day 6, the BMof recipient mice were analyzed by FACS and the number of hostphagocytic Gr1^(high) monocytes, Gr1^(low) monocytes, macrophages andneutrophils quantified. Error bars, mean±s.e.m. Unpaired Student's ttest (FIGS. 16A, 16C).

FIGS. 17A-17C. FIGS. 17A-17C depict experimental results showing thatthe Csf1r-iCre transgene is genetically linked to the MHC locus. FIG.17A depicts representative FACS plots of DAPI⁻ BMNCs cells from the sameH-2^(b/q) MHC-haplotype heterozygous mouse. Cells were stained withantibodies against MHC-I and II subclasses corresponding to the H-2^(b)(C57BL/6 strain) and H-2^(q) (FVB/N strain) haplotypes. FIG. 17B depictsa table showing calculation of the frequency of recombination betweenthe Csf1r-iCre transgene and the MHC locus. FIG. 17C depicts a series ofcharts showing quantification of tri-lineage (myeloid, B cell, and Tcell) engraftment in the blood of mice analyzed in FIG. 9C (n=6-14).Error bars, mean±s.e.m. Unpaired Student's t test (FIG. 17C).

FIGS. 18A-18C. FIGS. 18A-18C depict experimental results showing thesurvival defect of mice transplanted with VCAM1^(Csf1r-iCre); H-2^(b/q)BMNCs could not be rescued by CD8⁺ T cells depletion. Recipient micewere treated with a monoclonal anti-CD8 antibody or IgG control,lethally irradiated and transplanted with 1 million BMNCs fromhaplotype-mismatched control VCAM1⁻ or VCAM1^(Csf1r-iCre) mice (FIGS.18A, 18B). FIG. 18A depicts representative FACS plots and percentage ofT cell populations in the peripheral blood of mice treated with anti-CD8antibody or IgG control before transplantation. FIG. 18B depictssurvival curves of recipient mice depleted of CD8⁺ T cells and BMtransplanted (n=5). FIG. 18C depicts a chart showing quantification ofreconstituting HSPCs from syngeneic and haplotype mismatch VCAM1^(fl/fl)and VCAM1^(Csf1r-iCre) cells by competitive reconstitution assays intoPirb^(−/−) mice (n=6). Donor cell engraftment was evaluated by detectingthe levels of donor male DNA in the female recipient blood, by real-timePCR. Error bars, mean±s.e.m. Log-rank analysis was used for theKaplan-Meier survival curves in FIG. 28B. One-way ANOVA analysesfollowed by Tukey's multiple comparison tests was used in FIG. 18C.

FIGS. 19A-19E. FIGS. 19A-19E depict experimental results showing thatloss of VCAM1 inhibits the establishment and progression ofMLL-AF9-induced AML. FIG. 19A depicts a schematic overview ofexperimental strategy. FIG. 19B shows representative FACS plots of BMleukemic stem cells (LSCs) from control AML-VCAM1^(fl/fl) (top) andAML-VCAM1^(Csf1r-iCre) (bottom) primary recipients, 55 days aftertransplantation. FIG. 19C depicts a histogram showing the presence ofleukemic VCAM1⁺ LSCs derived from VCAM1^(Csf1r-iCre) mice in the BM ofmoribund secondary recipient mice, 103 days post-transplant. FIG. 19Ddepicts charts quantifying the number of phagocytic cells per femur inC57BL/6 mice treated with PBS or clodronate liposomes and transplantedwith control VCAM1^(fl/fl) or VCAM1^(Csf1r-iCre) pre-leukemic cells, atweek 4 (n=5). FIG. 19E depicts charts showing experimental resultswherein MLL-AF9-GFP⁺ cells were incubated in the presence of anti-VCAM1blocking antibody, isotype control or camptothecin-positive control.After 4.5 hours incubation, apoptotic cells were identified by Annexin Vstaining, as determined by FACS (n=4). Error bars, mean±s.e.m. One-wayANOVA analyses followed by Tukey's multiple comparison tests (FIGS. 19D,19E).

FIGS. 20A-20J. FIGS. 20A-20J depict experimental results showing thathigh VCAM1 expression correlates with poor prognosis in human AML. FIG.20A depicts a schematic overview of experiment strategy. MOLM-13 cellswere transduced with a human VCAM1-ZsGreen expressing (hVCAM1) orZsGreen control (Mock) lentivirus and transplanted intoimmunocompromised NODscid Il2rg^(−/−) (NSG) mice. FIG. 20B depicts ahistogram showing human VCAM1 expression on Mock⁻ and hVCAM1-MOLM-13cells. FIG. 20C depicts a chart showing percentage of human CD45⁺ AMLcells in the blood of MOLM-13 transplanted mice. FIG. 20D depictsKaplan-Meier survival analysis of mice receiving Mock− andhVCAM1-MOLM-13 cells. FIGS. 20E and 20F depict charts showing BMcellularity (FIG. 20E) and number of MOLM-13 cells that homed to the BMof recipient mice (FIG. 20F), 3 hours after injection. FIGS. 20G and 20Hdepict a chart showing the percentage of Annexin V⁺ apoptotic (FIG. 20G)and proliferating BrdU⁺ MOLM-13 cells (FIG. 20H) in the BM of recipientmice. FIG. 20I depicts the Kaplan-Meier survival analysis of mice withestablished hVCAM1-MOLM-13 AML (>1% human CD45⁺ cells in the blood) andtreated with daily injections of IgG1 (100 μg) and anti-human VCAM1monoclonal antibody (100 μg) for 10 days. FIG. 20J depicts a chartquantifying VCAM1 gene expression data from sorted human AML BM samplesand healthy controls. Lineage⁻ CD34⁺ CD38⁻ CD90⁺ cells (referred to asLT-HSCs), Lineage⁻ CD34⁺ CD38⁻ CD90⁻ cells (referred to as ST-HSCs), andLineage⁻ CD34⁺ CD38⁺ CD123⁺ CD45RA⁺ cells (referred to as GMPs).Cytogenetic abnormalities are depicted as: NK, normal karyotype; CK,complex karyotype; 7q, deletion of chromosome 7 (n=4-6). Error bars,mean±s.e.m. Unpaired student's t test (FIGS. 20C, 20E-20H). Log-rankanalysis was used for the Kaplan-Meier survival curves in FIGS. 20D, and20I. One-way ANOVA analyses followed by Tukey's multiple comparisontests (FIG. 20J). ns, non-significant.

FIGS. 21A-21G. FIGS. 21A-21G depict a series of experimental resultsshowing that deletion of MAEA impairs bone marrow macrophage developmentand the erythroblastic island. FIG. 21A depicts a representativehistogram showing MAEA expression on BM leukocytes from MAEA^(fl/fl)control and MAEA^(Csf1r-Cre) mice. FIG. 21B depicts a chart showingdeletion efficiency by Csf1r-Cre of MAEA on bone marrow macrophages asdetermined by FACS (n=8). FIG. 21C depicts a chart showingquantification of total BM cellularity in MAEA^(fl/fl) andMAEA^(Csf1r-Cre) mice (n=8). FIGS. 21D and 21E depict representativeFACS plots and quantification showing significant reduction ofmacrophage (FIG. 21D) and erythroblast (FIG. 21E) numbers in the bonemarrow of MAEA^(Csf1r-Cre) mice compared to littermate control (n=8).FIG. 21F is a chart depicting quantification of erythroblasts at variousstages of maturation (subpopulation I-V represents: I: proerythroblasts,II: basophilic erythroblasts, III: polychromatic erythroblasts, IV:orthochromatic erythroblasts and reticulocytes, V: mature RBCs) (n=8).FIG. 21G is a chart depicting RBC counts of MAEA^(fl/fl) andMAEA^(Csf1r-Cre) mice (n=10). Data are shown as mean±s.e.m. *p<0.05,**p<0.01, ***p<0.001, ****p<0.0001 by unpaired Student's t test.

FIGS. 22A-22H. FIGS. 22A-22H depict a series of experimental resultsshowing that deletion of MAEA impairs bone marrow macrophage developmentand erythroblastic niche. FIG. 22A depicts a schematic representation ofthe MAEA^(targeted) allele, MAEA^(floxed) allele and MAEA^(delta) allelegenerated using EuMMCR targeting vector PG00141_Z_1_G10. Exons aredepicted by boxes with coding regions indicated by shading. FRT sitesare marked as white triangles and LoxP sites as black triangles. TheIRES-LacZ reporter (LacZ) and the neomycin resistance cassette (Neo)were deleted by crossing with a Flpe-expressing deleter strain. Upontissue-specific or temporal Cre recombinase induction, the MAEA exon 5will be deleted which will result in a null MAEA^(delta) allele causedby non-sense mediated decay. FIG. 22B is an image of PCR analysisidentifying the wild-type (WT), MAEA^(floxed), and MAEA^(targeted)alleles. FIG. 22C depicts representative FACS plots and quantificationshowing impaired in vivo formation of BM erythroblastic islands (F4/80⁺Ter 19⁺ live multiplets) in MAEA^(Csf1r-Cre) mice (n=5). FIG. 22Ddepicts Wright-Giemsa stained smears from control and MAEA^(Csf1r-Cre)peripheral blood. Scale bar=50 μm. FIG. 22E depicts a chart showingquantification of spleen erythroblasts in MAEA^(Csf1r-Cre) mice (n=8).FIG. 22F depicts a chart showing burst-forming unit-erythroid (BFU-E) inMAEA^(Csf1r-Cre) spleen (n=5). FIG. 22G depicts representativehistograms and quantification shown prolonged half-life of in vivobiotinylated RBCs in MAEA^(Csf1r-Cre) mice (n=5). FIG. 22H depictscharts showing RBC counts and HCT in splenectomized control andMAEA^(Csf1r-Cre) mice (n=5). Data are shown as mean±s.e.m. *p<0.05,**p<0.01, ***p<0.001, ****p<0.0001 by unpaired Student's t test.

FIGS. 23A-23H. FIGS. 23A-23H depict experimental results showing thatMAEA is required for HSC engraftment. FIG. 23A depicts a series ofcharts showing the reconstitution capability of MAEA^(fl/fl) andMAEA^(Csf1r-Cre) HSCs as determined by competitive BM transplantation(n=5). 1×10⁶ of donor (CD45.2) BM cells were competitively transplantedwith equal number of competitor (CD45.1) BM cells into lethallyirradiated recipient mice (CD45.1). Percentage of donor derived B220⁺ B,CD3⁺ T, and CD11b⁺Gr1⁺ myeloid cells were quantified at indicated timepoints. FIG. 23B is a schematic diagram of the experimental design ofthe reciprocal BMT performed. FIG. 23C depicts a chart showing thepercentage of donor derived cells were quantified in the BM, peripheralblood, and spleen of the control and MAEA^(Csf1r-Cre) recipients 16weeks after the transplant. FIGS. 23D and 23E depict charts quantifyingWBC counts (FIG. 23D) and BM cellularity (FIG. 23E) in control andMAEA^(Csf1r-Cre) recipient mice 16 weeks after the transplant (n=5).FIG. 23F depicts a chart quantifying the frequency of B, T, and myeloidcells in total WBCs of control and MAEA^(Csf1r-Cre) reciprocalrecipients (n=5). FIGS. 23G and 23H depict charts showing thequantification of BM macrophages (FIG. 23G) and erythroblasts (FIG. 23H)control and MAEA^(Csf1r-Cre) reciprocal recipients (n=5). Data are shownas mean±s.e.m.

FIGS. 24A-24F. FIGS. 24A-24F depict experimental results demonstratingthat MAEA is required for HSC engraftment but dispensable for theirmaintenance. FIG. 24A depicts plots of cell cycle analysis of BM HSCs byKi-67 and H33342 dye staining (n=3). FIG. 24B depicts charts quantifyingcleaved caspase3 expression in BM LSKs from the control andMAEA^(Csf1r-Cre) mice (n=3). FIG. 24C depicts charts showing anassessment of peripheral blood recovery of the control andMAEA^(Csf1r-Cre) mice after 250 mg/kg of 5-FU challenge (n=6). FIG. 24Ddepicts charts showing BM total cellularity, LSK, and HSC numbers of thecontrol and MAEA^(Csf1r-Cre) mice 4 weeks after 5-FU injection. FIG. 24Edepicts charts showing the quantification of homed BMNCs and LK cellsfrom control and MAEA^(Csf1r-Cre) mice in lethally irradiated WT CD45.1recipients (n=5). FIG. 24F depicts a chart showing comparabledifferentiation potential of control and MAEA^(Csf1r-Cre) LSK cellsmeasured by colony-forming assays (n=4).

FIGS. 25A-25D. FIGS. 25A-25D depict experimental results demonstratingthat MAEA over-expression is associated with poor prognosis of humancancers. FIG. 25A depicts a chart showing cross-cancer alterationsummary for MAEA from 126 human cancer genomics studies generated bycBioPortal for Cancer Genomics from MSKCC. FIG. 25B depicts chartsshowing Kaplan-Meier overall and disease-free survival of AML patients(TCGA, NEJM 2013) with high and low MAEA expression (mRNA expressionz-Score threshold±2). FIGS. 25C and 25D depict charts showingKaplan-Meier overall survival of ovarian cancer and lung adenocarcinomapatients (TCGA) with high and low MAEA expression (mRNA expressionz-Score threshold±2). The significance is based on log rank testestimate of p values.

FIGS. 26A-26K. FIGS. 26A-26K depict experimental results demonstratingthat MAEA is required for mouse AML engraftment and progression. FIG.26A depicts a schematic showing development of an MLL-AF9 acute myeloidleukemia (AML) model. FIG. 26B depicts a chart showing expression levelof MAEA, measured by mean fluorescent intensity (MFI), in total bonemarrow cells (BM), LSK, lineage⁻ ckit⁺ (LK) and granulocyte-macrophageprogenitors (GMP) of healthy and AML mice. In leukemic mice, both GFP⁺AML cells (AML) and their residual GFP⁻ healthy counterparts (non-AML)were assessed. FIG. 26C depicts a chart quantifying of GFP⁺ AML cells inprimary sub-lethally irradiated recipients that received Ctrl andMAEA^(Csf1r-Cre−) pre-leukemic cells. FIG. 26D depicts charts showing anassessment of the peripheral blood of mice transplanted with control andMAEA^(Csf1r-Cre) pre-leukemic cells at 10-12 weeks after transplant(PLT=platelet). FIG. 26E depicts a survival curve of mice transplantedwith control and MAEA^(Csf1r-Cre) pre-leukemic cells (n=5). p value isdetermined by Log-rank test. FIG. 26F depicts representative FACSanalysis of BM cells from control and MAEA^(Csf1r-Cre) pre-leukemic miceat 10-12 weeks after transplant. FIGS. 26G and 26H depict charts showingquantification of total leukaemia load (GFP⁺) (FIG. 26G) and leukemicGMP (L-GMP) (FIG. 26H) in recipients of control (black) andMAEA^(Csf1r-Cre) (grey) pre-leukemic cells at 10-12 weeks aftertransplant (n=5). FIG. 26I depicts a chart showing the progression ofcirculating control and MAEA^(Mx1-Cre) AML cells after a singleinjection of pIpC (arrow). FIG. 26J depicts a chart showing theprogression of circulating wild type AML cells after injections of 60 μganti-MAEA polyclonal antibody (arrows). FIG. 26K depicts a schematic ofexperimental design and a Kaplan-Meyer survival curve of wildtype micetransplanted with MLL-AF9 leukemic BM cells treated with IgG oranti-MAEA monoclonal antibody (92.25) after establishment of theleukaemia (n=5). Data are shown as mean±s.e.m. *p<0.05, **p<0.01,***p<0.001, ****p<0.0001 by unpaired Student's t test.

FIGS. 27A-27E. FIGS. 27A-27E depict experimental results of wild typemice treated with IgG and anti-MAEA antibody. Wild type mice were giventhree doses of 60 μg IgG and anti-MAEA antibody i.v. every other day andanalysed 2 days after the last injection. FIGS. 27A-27D depict a seriesof charts showing total body, spleen, and liver weight (FIG. 27A), BMand spleen cellularity (FIG. 27B), erythroblasts percentage in the BM(FIG. 27C), and LSK and HSC percentages in the BM and spleen (FIG. 27D)of IgG and anti-MAEA antibody treated mice. FIG. 27E depicts a tablesummary of peripheral blood parameters from mice treated with IgG andanti-MAEA antibody. (WBC=white blood cells; RBC=red blood cells;HGB=haemoglobin; HCT=haematocrit; MCV=mean corpuscular volume;PLT=platelets; Retic=reticulocyte; Lymph=lymphocyte). Data are shown asmean±s.e.m. *p<0.05, **p<0.01 by unpaired Student's t test.

FIGS. 28A-28D. FIGS. 28A-28D depict experimental results showing MAEAexpression in human cancer cell lines. RNA-seq data of 675 human cancercell lines across tissue types were previously published⁴⁵ and madeavailable on the web at research-pub.gene.com/KlijnEtAl2014. FIG. 28Adepicts a chart showing the distribution of MAEA mRNA expression (RPKM)across all 675 lines. FIG. 28B depicts a chart showing MAEA expressionin cancer cell lines across their tissue origin. FIGS. 28C and 28Ddepict charts showing MAEA expression in lung (FIG. 28C) and ovarian(FIG. 28D) cancer cell lines.

FIGS. 29A-29F. FIGS. 29A-29F depict experimental results showing thatMAEA maintains an adult BM erythroblastic island niche. FIG. 29A depictsa chart showing validation of the specificity of mAb produced byhybridoma clone 92.25 by FACS staining of BM macrophages from wild typecontrol and MAEA^(Csf1r-Cre) mice. FIGS. 29B-29E depict charts showingquantification of total BM cellularity (FIG. 29B), BM EB numbers (FIG.29C), EB maturation profile (FIG. 29D), and BM macrophage numbers (FIG.29E) in isotype or anti-MAEA mAb-treated mice (n=5). FIG. 29F depicts achart showing quantification of erythroid cells per erythroblasticisland reconstituted in the presence of 10 μg/mL isotype or anti-MAEAmAb. Data are shown as mean±s.e.m. *p<0.05, **p<0.01, ****p<0.0001 byunpaired Student's t test.

FIGS. 30A-30H. FIGS. 30A-30J depict experimental results demonstratingthat MAEA expression is enriched and required in HSCs for hematopoiesis.FIG. 30A depicts representative histograms showing MAEA expression onLSK and HSCs in control and MAEA^(Csf1r-Cre) BM. FIG. 30B depicts achart showing FACS quantification of MAEA expression on control andMAEA^(Csf1r-Cre) BM HSPCs (LMPP=lymphoid-primed multipotent progenitors;CMP=common myeloid progenitors; CLP=common lymphoid progenitors;GMP=granulocyte-macrophage progenitors; MEP=megakaryocyte-erythrocyteprogenitors; neutrophils=Neu; B cells=B; and monocytes=MN) (n=3 eachgroup). FIG. 30C depicts a chart showing the Kaplan-Meyer survival curveof control and MAEA^(Csf1r-Cre) mice. p value was calculated by log-ranktest (n=5-8). FIG. 30D depicts charts showing WBC counts in peripheralblood and frequency of B, T and myeloid cells in total WBCs of youngadult control and MAEA^(Csf1r-Cre) mice. FIG. 30E depicts charts showingquantifications of HSC and LSK numbers in BM of control andMAEA^(Csf1r-Cre) mice. FIG. 30F depicts charts showing quantification ofmyeloid progenitors in BM of control and MAEA^(Csf1r-Cre) mice. FIG. 30Gdepicts charts showing quantification of lymphoid progenitors in BM ofcontrol and MAEA^(Csf1r-Cre) mice. FIG. 30H depicts a schematic of anexperimental scheme and charts depicting results for evaluating lymphoiddifferentiation potential of HSC and LMPP at single cell level. Data areshown as mean±s.e.m. *p<0.05 by unpaired Student's t test.

FIGS. 31A-31K. FIGS. 31A-31K depict experimental results demonstratingthat an MAEA-deletion impairs HSC quiescence and function in amTOR-dependent manner. FIG. 31A depicts a schematic diagram of anexperimental scheme for deleting MAEA in adult mice using Mx1-Cre. FIG.31B depicts a chart showing quantification of BM HSC numbers atindicated time points after poly I:C injection. FIG. 31C depictsrepresentative FACS plots and cell cycle profiles of control andMAEA^(Mx1-Cre) HSCs at 21 days after a first poly I:C injection. FIG.31D depicts a schematic of an experimental scheme for deleting MAEA in1:1 wild type and MAEA^(Mx1-Cre) BM chimeras after stable (8 weeks)reconstitution. FIG. 31E depicts a series of charts showing donorchimerism in BM LSK and HSCs and peripheral blood total leukocytes fromcontrol and MAEA^(Mx1-Cre) mixed chimeric mice at indicated time pointsafter poly I:C injection. FIG. 31F depicts RNA-seq and Gene SetEnrichment Analysis (GSEA) of control and MAEA^(Csf1r-Cre) HSCs. Threereplicates with 2000 HSCs were pooled from 2 mice each replicate wereprocessed and analyzed for each group. Top KEGG (Kyoto Encyclopedia ofGenes and Genomes) pathways that are up-regulated and downregulated inMAEA^(Csf1r-Cre) HSCs are shown. FIG. 31G depicts three examples of GSEAenrichment plots showing enrichment of Proteasome, Oxidativephosphorylation, and mTOR signaling pathways in MAEA^(Csf1r-Cre) HSCs.FIG. 31H depicts a GSEA enrichment plot showing significantdown-regulation of lymphoid potential related gene set inMAEA^(Csf1r-Cre) HSCs. FIG. 31I depicts a heat map showing meanexpression of HSC related genes in control and MAEA^(Csf1r-Cre) HSCs.FIG. 31J depicts a schematic representation of an experimental schemeand a chart quantifying LSKs and HSCs in control and MAEA^(Mx1-Cre) micetreated with vehicle, carfilzomib, rapamycin, or NAC 3 weeks after polyI:C induction. FIG. 31K depicts a chart showing donor chimerism inperipheral blood of CD45.1 lethally irradiated wild-type recipients atindicated time points after competitive BM transplantation of equalnumber of CD45.1 wild type competitor BM cells and CD45.2 donor BM cellsfrom indicated groups (n=5 each group). Data are mean±s.e.m. *p<0.05,**p<0.01, ***p<0.001, ****p<0.0001 by unpaired Student's t test. n.s.,not significant.

FIGS. 32A-32I. FIGS. 32A-32I depict experimental results demonstratingthat MAEA regulates cytokine receptor stability and autophagy in HSCs.FIG. 32A depicts representative immunofluorescence images showing MAEAand CD150 expression in HSCs (scale bar=10 m). FIG. 32B depictsrepresentative ubiquitin array images and quantified mean spot pixelintensity of selected targets from freshly isolated control andMAEA^(Csf1r-Cre) lineage negative BM cells (n=5). *p<0.05, **p<0.01,***p<0.001, ****p<0.0001 by unpaired Student's t test. FIG. 32C depictsrepresentative histograms and FACS evaluation of Flt3 half-life incontrol and MAEA^(Csf1r-Cre) LSK cells incubated in the presence of 50μM cycloheximide (n=4). *p<0.05, **p<0.01, ***p<0.001 by two-way ANOVAmultiple comparisons. FIG. 32D depicts a schematic overview of anexperimental scheme and evaluation of autophagy flux in control andMAEA^(Csf1r-Cre) HSCs. Percent autophagy flux is calculated as100×(1−(−L/N))/(+L/N). N: NH4Cl. L: leupeptin. FIG. 32E depictsrepresentative EM micrographs of control and MAEA^(Csf1r-Cre) HSCs forultrastructural analysis of the autophagic compartments (arrows point toan autolysosome in control and an autophagosome in MAEA^(Csf1r-Cre)).FIG. 32F depicts charts showing morphometric analysis of control andMAEA^(Csf1r-Cre) HSCs: quantification of the numbers of autophagicvacuoles (AV) and their break down into number (left) and percentage(right) of autophagosomes (APG) and autolysosomes (AUT), per cell area.At least 20 cells were analyzed in each group. FIG. 32G depicts a chartshowing frequencies of HSCs in control and MAEA^(Csf1r-Cre) BM cellsbefore and after 3 hours of starvation in culture. FIG. 32H depictscharts showing quantification of LSKs and HSCs in control andMAEA^(Mx1-Cre) BM treated with vehicle, lithium chloride, or verapamil 3weeks after poly I:C induction. FIG. 32I depicts a chart showing overallperipheral blood donor chimerism in CD45.1 lethally irradiated wild typerecipients at indicated time points after competitive BM transplantationof equal number of CD45.1 wild type competitor BM cells and CD45.2 donorBM cells from indicated groups (n=5 each group). Data are shown asmean±s.e.m. n.s., not significant. *p<0.05, **p<0.01, ***p<0.001,****p<0.0001 by unpaired Student's t test unless otherwise indicated.

FIGS. 33A-33D. FIGS. 33A-33D depict experimental results demonstratingthat MAEA^(Csf1r-Cre) mice develop myeloproliferation and lymphopenia.FIG. 33A is a series of charts showing peripheral blood indices of 7months old control and MAEA^(Csf1r-Cre) mice. FIG. 33B depictsrepresentative H&E stained paraffin sections from the livers and lungsof 7 months old control and MAEA^(Csf1r-Cre) mice. Scale bar=50 μm.FIGS. 33C and 33D depict representative FACS plots (FIG. 33C) andquantification (FIG. 33D) of BM cellularity, Gr-1⁺ and B220⁺ cells intotal BM nucleated cells in 7 months old control and MAEA^(Csf1r-Cre)mice. Data are shown as mean±s.e.m. *p<0.05, **p<0.01, ***p<0.001,****p<0.0001 by unpaired Student's t test.

FIGS. 34A-34E. FIGS. 34A-34E depict experimental results showing alteredhematopoiesis in MAEA^(Csf1r-Cre) mice. FIG. 34A depicts representativeFACS plots and quantifications showing increased LSK, LK, and HSCs inthe MAEA^(Csf1r-Cre) mice. Events are pre-gated on the lineage⁻populations. FIG. 34B depicts a representative gating strategy for GMP,CMP, MEP, and MkP from the LK population in FIG. 34A. FIG. 34C depictsrepresentative FACS plots showing decreased frequencies of LMPPs in theLSK population from the MAEA^(Csf1r-Cre) mice. FIG. 34D depictsrepresentative FACS plots showing decreased frequencies of CLPs in theLin-Sca1^(lo)ckit^(lo) population from the MAEA^(Csf1r-Cre) mice. FIG.34E depicts charts showing homed control and MAEA^(Csf1r-Cre) total BMcells and ckit⁺ cells in recipient BM 3 hours after transplantation (n=5each group). Data are shown as mean±s.e.m. **p<0.01, ***p<0.001,****p<0.0001 by unpaired Student's t test.

FIGS. 35A-35E. FIGS. 35A-35E depict experimental results demonstratingthat MAEA-deletion impairs HSC quiescence and function. FIG. 35A depictsa chart quantifying LSK numbers per femur in control and MAEA^(Mx1-Cre)BM at indicated time points after 1st poly I:C injection. FIG. 35Bdepicts a chart quantifying BM cellularity of control and MAEA^(Mx1-Cre)mice at indicated time points after 1st poly I:C injection. FIG. 35Cdepicts quantifying cell cycle profiles of control and MAEA^(Mx1-Cre)total lineage⁺ and lineage⁻ cells at 21 days after 1st poly I:Cinjection. FIG. 35D depicts a chart quantifying cell cycle analysis ofBM HSCs from control and MAEA^(Csf1r-Cre) mice by Ki-67 and H33342 dyestaining (n=4). FIG. 35E depicts a chart quantifying HSC numbers in7-month-old control and MAEA^(Csf1r-Cre) BM. Data are shown asmean±s.e.m. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by unpairedStudent's t test.

FIGS. 36A-36G. FIGS. 36A-36G show experimental results demonstratingthat deletion of MAEA results in aberrant HSC activation and depletionin a mTOR-dependent manner. FIG. 36A depicts a GSEA enrichment plotshowing significant upregulation of DNA replication pathway (left) anddown-regulation of insulin signalling pathway (right) inMAEA^(Csf1r-Cre) HSCs. FIG. 36B depicts representative histogramsshowing total S6 and pS6 levels and pS6/S6 ratio in control andMAEA^(Csf1r-Cre) HSCs. FIG. 36C depicts a pair of charts showing pAKT(S473) and pERK1/2 (p44/42 MAPK, T202/Y204) levels in control andMAEA^(Csf1r-Cre) HSCs. FIGS. 36D and 36E depict charts showingquantification of BM cellularity (FIG. 36D) and BM LSK numbers (FIG.36E) in control and MAEA^(Mx1-Cre) mice treated with vehicle,carfilzomib, rapamycin, or NAC 3 weeks after poly I:C induction. FIG.36F depicts a series of charts showing donor chimerism in peripheralblood of CD45.1 lethally irradiated wild type recipients at theindicated time points after competitive BM transplantation of equalnumber of CD45.1 wild type competitor BM cells and CD45.2 donor BM cellsfrom indicated groups (n=5 each group). FIG. 36G depicts a chart showingquantification of BM macrophage numbers in control and MAEA^(Mx1-Cre)mice treated with vehicle, carfilzomib, rapamycin, or NAC 3 weeks afterpoly I:C induction. Data are shown as mean±s.e.m. n.s., not significant.*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by unpaired Student's ttest.

FIGS. 37A, 37B. FIGS. 37A and 37B depict experimental results showingthat MAEA regulates receptor stability and signaling. FIG. 37A is a pairof charts depicting a FACS evaluation of surface receptor Mpl and ckithalf-lives in control and MAEA^(Csf1r-Cre) HSPCs incubated in thepresence of 50 μM cycloheximide (n=4). FIG. 37B is a series of chartsdepicting phospho-flow evaluation of signaling molecules pAkt, pErk andpS6/S6 ratio in control and MAEA^(Csf1r-Cre) HSPCs after cytokine (TPO,FLT3, and SCF 20 ng/ml each) stimulation. Data in 37A and 37B are shownas mean±s.e.m. *p<0.05, ****p<0.0001 by two-way ANOVA multiplecomparisons.

FIGS. 38A-38F. FIGS. 38A-38F depict experimental results demonstratingthat MAEA regulates autophagy in HSCs. FIG. 38A is a chart showingexpression of core autophagy machinery in control and MAEA^(Csf1r-Cre)HSCs from RNA-seq analysis. FIG. 38B is a chart showing expression ofpro-autophagy genes in control and MAEA^(Csf1r-Cre) HSCs from RNA-seqanalysis. FIG. 38C depicts representative histograms and FACSquantifications showing autophagy flux by LC3-II in control andMAEA^(Csf1r-Cre) lineage⁺ (upper) and lineage⁻ (lower) BM cells,measured at the same time as in FIG. 33C. FIG. 38D depicts micrographsof whole cells (left) and examples of autophagosomes (APG, yellowarrows) and autolysosomes (AUT, red arrows) from control andMAEA^(Csf1r-Cre) HSCs. Bars in inserts=0.1 mm. FIG. 38E depicts a chartshowing quantification of BM macrophages in control and MAEA^(Mx1-Cre)mice treated with vehicle, lithium chloride, or verapamil 3 weeks afterpoly I:C induction. FIG. 38F depicts a series of charts showing donorchimerism in peripheral blood of CD45.1 lethally irradiated wild typerecipients at indicated time points after competitive BM transplantationof equal number of CD45.1 wild type competitor BM cells and CD45.2 donorBM cells from indicated groups (n=5 each group). Data are shown asmean±s.e.m. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by unpairedStudent's t test.

FIGS. 39A-39G. FIGS. 39A-390G depict experimental results showing theeffect of a VCAM-1 antibody on sickle cell disease (SCD) mice suggestingthat VCAM1 works via inhibition of leukocyte activation. FIG. 39Adepicts a schematic overview of experimental design. Mice were injectedby intravenous with 200 μg/mice rat IgG1 or VCAM1 antibody at 16 hoursand 2 hours before TNF-α challenge (n=5, rat IgG1 antibody 200 μg/miceor VCAM1 antibody 200 μg/mice). FIG. 39B depicts a chart showing whiteblood cell (WBC) rolling in mice injected with rat IgG or VCAM1 antibody(p<0.0001). FIG. 39C depicts a chart quantifying number of adhesions per100 μm in mice injected with rat IgG1 or VCAM1 antibody (p<0.0001). FIG.39D depicts a chart quantifying red blood cell (RBC) interactions perWBCs in mice injected with rat IgG or VCAM1 antibody (p<0.05). FIG. 39Edepicts extravasated WBCs in mice injected with rat IgG1 or VCAM1antibody (n.s.). FIG. 39F depicts a chart showing a survival curve formice injected with rat IgG or VCAM1 antibody. FIG. 39G depicts a tableshowing number of venules, venule diameter, centerline velocity, shearrate, and blood flow in mice injected with rat IgG1 or VCAM1 antibody.

FIGS. 40A-40D. FIGS. 40A-40D depict experimental results quantifyingwhite blood cell numbers and types in mice injected with rat IgG1 orVCAM1 antibody. FIG. 40A depicts a chart quantifying WBCs in miceinjected with rat IgG1 or VCAM1 antibody (p<0.05). FIG. 40B depicts achart quantifying the blood differential count (by percent) in miceinjected with rat IgG1 or VCAM1 antibody (eosinophils=white;lymphocytes=grey; neutrophils=black). FIG. 40C depicts a chartquantifying the blood cell count in mice injected with rat IgG1 or VCAM1antibody (eosinophils=white; lymphocytes=grey; neutrophils=black). FIG.40D is a chart depicting serum inflammatory cytokine levels (pg/ml) inmice injected with rat IgG1 or VCAM1 antibody.

FIG. 41 . FIG. 41 depicts a chart showing data indicating that the VCAM1receptor CD49d is expressed on mouse neutrophils from SA and SS SCDmice.

FIGS. 42A-42C. FIGS. 42A-42C depict experimental results demonstratingthat anti-MAEA monoclonal antibody treatment benefitsJak2^(V617F)-induced (R/R) Polycythemia Vera (PV). FIG. 42A depicts aseries of charts quantifying complete blood count (CBC) results showingthat anti-MAEA monoclonal antibody (92.25) injections lowered thereticulocytes, red blood cell counts (RBC), and haemoglobin levels (HGB)in the peripheral blood of Jak2^(R/R) mice without affecting the controlmice. FIG. 42B depicts a series of charts quantifying erythroblast (EB)numbers in bone marrow (BM) following anti-MAEA monoclonal antibody(92.25) injections. EB numbers were reduced by 92.25 injections in bothcontrol and Jak2^(R/R) mice relative to IgG control groups, whilemacrophage numbers were only reduced in the Jak2^(R/R) mice. FIG. 42Cdepicts a pair of charts showing anti-MAEA monoclonal antibody (92.25)injections enhanced the maturation of EBs in the BM of Jak2^(R/R) micewithout altering the EB maturation profile in wild type (WT) controlmice.

DETAILED DESCRIPTION

The following description of the invention is merely intended toillustrate various embodiments of the invention. As such, the specificmodifications discussed are not to be construed as limitations on thescope of the invention. It will be apparent to one skilled in the artthat various equivalents, changes, and modifications may be made withoutdeparting from the scope of the invention, and it is understood thatsuch equivalent embodiments are to be included herein.

Haematopoietic stem cells (HSCs) home to the bone marrow (BM) via, inpart, the interactions with vascular cell adhesion molecule-1(VCAM1).⁵⁷⁻⁵⁹ Upon migrating into the BM, HSCs are vetted byperivascular phagocytes to ensure their self-integrity. As set forth inthe experimental results provided herein, VCAM1 is also expressed onhealthy HSCs and upregulated on leukemic stem cells (LSCs) where itserves as a quality-control checkpoint for entry into BM by providing‘don't-eat-me’ stamping in the context of major histocompatibilitycomplex (MHC) class-I presentation. The results provided herein suggestthat VCAM1 engagement regulates a critical immune checkpoint gate in theBM and offers a novel strategy to eliminate cancer cells via modulationof the innate immune tolerance.

Sickle cell disease (SCD) is characterized by sickle-shaped RBCs, whichexhibit increased adherence to other blood cells or to the endothelium,and are prone to undergo premature clearance and hemolysis.⁴⁸ The RBCalterations lead to a chronic inflammatory state resulting in ischemictissue damage manifested by severe pain and organ failures. Thepathophysiology of VOE and chronic organ damage is complex and involvesthe interplay of altered blood rheology, endothelial activation, and thesecretion of inflammatory cytokines enabling leukocyte adhesion andactivation.⁴⁹ Intravital microscopy analysis of the SCD mousemicrocirculation has revealed that RBCs interact with adherentleukocytes in the inflamed vasculature.⁵⁰ The accumulation of activatedneutrophils interacting with RBCs progressively reduces blood flow andproduces venular occlusions.^(51,52) Results provided herein indicatethat blocking VCAM1 function may help treat SCD by reducing the numberof WBC adhesions, reducing the strength for rolling interactions(increased the number of rolling WBCs), reducing RBC/WBC interactions,increasing centerline velocity, decreasing shear rate, and prolongingsurvival.

Macrophage-Erythroblast Attacher (MAEA, also known as EMP) wasoriginally identified as an adhesion molecule required forerythroblastic island formation.⁷ Germline deletion of MAEA led tosevere anemia and perinatal mortality.⁹⁷ Sequence analysis indicatesthat MAEA is a highly conserved RING finger domain-containing E3ubiquitin ligase.^(98,99) MAEA's functions, however, remain obscure. Asshown by results provided herein, MAEA is highly expressed inhematopoietic stem cells (HSCs) where it is required for theirmaintenance by restricting cytokine receptor signaling and promotingautophagy. Constitutive MAEA deletion produces severe defects in HSCrepopulation capacity, B- and T-lymphoid differentiation, and prematuredeath of animals from a myeloproliferative syndrome. Postnatal MAEAdeletion leads to transient HSC expansion followed by their depletion.Mechanistically, Applicants found that the surface expression of severalhematopoietic cytokine receptors (e.g., MPL, FLT3) is stabilized inabsence of MAEA, thereby prolonging their intracellular signaling.Additionally, the autophagy flux in HSCs, but not in maturehematopoietic cells, is markedly impaired. Administration ofautophagy-inducing compounds rescued the functional defects ofMAEA-deficient HSCs. Further, MAEA is upregulated in various cancers andassociated with poor survival of acute myelogenous leukemia (AML), andMLL-AF9-driven AML does not develop in the absence of MAEA. Moreover,treatment of AML-bearing mice with an anti-MAEA antibody significantlyimproved their survival.

Antibodies and antibody fragments that inhibit the activity of vascularcell adhesion molecule 1 (VCAM1) and/or macrophage erythroblast attacher(MAEA) are provided, along with formulations and kits comprising theseantibodies and antibody fragments. Also provided are methods oftreatment using the disclosed compositions, formulations, and kits totreat conditions such as cancers, sickle cell disease (SCD), andPolycythemia Vera. Methods of treating SCD by blocking VCAM1 areprovided based on the surprising findings disclosed herein.

Provided herein are VCAM1 inhibitors and MAEA inhibitors, e.g., VCAM1 orMAEA antibodies, and formulations and kits comprising those inhibitors.Also provided herein are methods for treating Polycthemia Vera (PV) byblocking VCAM1 or MAEA using a VCAM1 inhibitor or MAEA inhibitor, e.g.,by using one of the antibodies of the present disclosure. Furtherprovided are methods for treating sickle cell disease (SCD) by blockingVCAM1 using a VCAM inhibitor, e.g., by using one of the antibodies ofthe present disclosure.

A “VCAM1 inhibitor” as used herein refers to any molecule that inhibitsthe activity of VCAM1, either partially or completely. An “MAEAinhibitor” as used herein refers to any molecule that inhibits theactivity of MAEA, either partially or completely.

In certain embodiments of the methods, compositions, and kits providedherein, the VCAM1 and/or MAEA inhibitor inhibits VCAM1 and/or MAEA bybinding to VCAM1 and/or MAEA. Examples of such VCAM1 and/or MAEAinhibitors include, e.g., antagonistic VCAM1 and/or MAEA antibodies orfusion proteins thereof, inactive forms of a VCAM1 and/or MAEA ligand(e.g., a truncated or otherwise mutated form of a VCAM1 and/or MAEAligand) or fusion proteins thereof, small molecules, siRNAs, oraptamers.

In certain preferred embodiments of the methods, compositions, and kitsprovided herein, the antibody or antibody fragment is an antagonistic orblocking antibody or fragment. The antibody or antibody fragment thatspecifically inhibits the activity of VCAM1 is preferably a blockingantibody to VCAM1 or an antibody fragment that blocks the activity ofVCAM1. The antibody or antibody fragment that specifically inhibits theactivity of MAEA is preferably a blocking antibody to MAEA or anantibody fragment that blocks the activity of MAEA. As used herein, theterm “antibody” refers to an intact antibody, i.e., with complete Fc andFv regions. Antibody “fragment” refers to any portion of an antibody, orportions of an antibody linked together, such as, in non-limitingexamples, a Fab, F(ab)₂, a single-chain Fv (scFv), which is less thanthe whole antibody but which is an antigen-binding portion and whichcompetes with the intact antibody of which it is a fragment for specificbinding to the target. As such a fragment can be prepared, for example,by cleaving an intact antibody or by recombinant means. See generally,Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y.(1989), hereby incorporated by reference in its entirety).Antigen-binding fragments may be produced by recombinant DNA techniquesor by enzymatic or chemical cleavage of intact antibodies or bymolecular biology techniques. In some embodiments, a fragment is an Fab,Fab′, F(ab′)2, Fd, Fv, complementarity determining region (CDR)fragment, single-chain antibody (scFv), (a variable domain light chain(VL) and a variable domain heavy chain (VH) linked via a peptide linker.In an embodiment, the scFv comprises a variable domain frameworksequence having a sequence identical to a human variable domain FR1,FR2, FR3 or FR4. In an embodiment, the scFv comprises a linker peptidefrom 5 to 30 amino acid residues long. In an embodiment, the scFvcomprises a linker peptide comprising one or more of glycine, serine andthreonine residues.

The “variable region” or “variable domain” of an antibody refers to theamino-terminal domains of the heavy or light chain of the antibody. Thevariable domain of the heavy chain may be referred to as “VH.” Thevariable domain of the light chain may be referred to as “VL.” Thesedomains are generally the most variable parts of an antibody and containthe antigen-binding sites. The term “variable” refers to the fact thatcertain portions of the variable domains differ extensively in sequenceamong antibodies and are used in the binding and specificity of eachparticular antibody for its particular antigen. However, the variabilityis not evenly distributed throughout the variable domains of antibodies.It is concentrated in three segments called hypervariable regions (HVRs)(or CDRs) both in the light-chain and the heavy-chain variable domains.The more highly conserved portions of variable domains are called theframework regions (FR). The variable domains of native heavy and lightchains each comprise four FR regions, largely adopting a beta-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the beta-sheet structure. The CDRs ineach chain are held together in close proximity by the FR regions and,with the CDRs from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al., Sequences ofProteins of Immunological Interest, Fifth Edition, National Institute ofHealth, Bethesda, Md. (1991)). The constant domains are not involveddirectly in the binding of an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (γ), based on the amino acid sequences of theirconstant domains.

“Framework” or “FR” residues are those variable domain residues otherthan the HVR residues as herein defined.

The term “hypervariable region” or “HVR” when used herein refers to theregions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six HVRs; three in the VH (H1, H2, H3) and three in the VL (L1,L2, L3).

Also provided is an Anti-MAEA mAb 92.25 having the following sequences,where the Signal sequence/peptide is in italics, CDRs 1-3 areunderlined, and the framework (FR) regions are not italicized orunderlined.

Heavy chain: DNA sequence (417 bp) Signal sequence-FR1- CDR1 -FR2- CDR2-FR3- CDR3 -FR4 (SEQ ID NO: 8)ATGGAATGGAGCTGGATCTTTCTCTTTCTCCTGTCAGGAACTGCAGGTGTCCTCTCTGAGGTCCAGCTGCAACAGTTTGGAGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACATTCACT GACTACAACATGGAC TGGGTGAAGCAGAGCCATGCAAAGAGACTTGAGTGGATTGGA GATATTAATCCTAACTATGATAGTCCTACCTACAGCCAGAA GTTCAAGGGAAGGGCCACATTGACTGTAGACAACTCCTCCAGCACCGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACACTGCAGTCTATTACTGTGCAAGG GGACATTACTACGGCTACGGATACTTCGATGTC TGGGGCGCGGGGACCACGGTCACCGTCTCCTCA;Heavy chain FR1: DNA sequence (SEQ ID NO: 20)GTCCAGCTGCAACAGTTTGGAGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACATTCACT; Heavy chain CDR1: DNA sequence(SEQ ID NO: 21) GACTACAACATGGAC; Heavy chain FR2: DNA sequence(SEQ ID NO: 22) TGGGTGAAGCAGAGCCATGCAAAGAGACTTGAGTGGATTGGAHeavy chain CDR2: DNA sequence (SEQ ID NO: 23)GATATTAATCCTAACTATGATAGTCCTACCTACAGCCAGAAGTTCAAGGGA;Heavy chain FR3: DNA sequence (SEQ ID NO: 24)AGGGCCACATTGACTGTAGACAACTCCTCCAGCACCGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACACTGCAGTCTATTACTGTGCAAGG; Heavy chain CDR3: DNA sequence(SEQ ID NO: 25) GGACATTACTACGGCTACGGATACTTCGATGTC;Heavy chain FR4: DNA sequence (SEQ ID NO: 26)TGGGGCGCGGGGACCACGGTCACCGTCTCCTCA;Heavy chain: Amino acid sequence (139 aa) Signal peptide-FR1- CDR1 -FR2-CDR2 -FR3- CDR3 -FR4 (SEQ ID NO: 9)MEWSWIFLFLLSGTAGVLSEVQLQQFGAELVRPGASVKISCKASGYTFT DYNMD WVKQSHAKR LEWIGDINPNYDSPTYSQKFKG RATLTVDNSSSTAYMELRSLTSEDTAVYYCAR GHYYGYGY FDVWGAGTTVTVSS; Heavy chain FR1: Amino acid sequence (SEQ ID NO: 27)EVQLQQFGAELVRPGASVKISCKASGYTFT; Heavy chain CDR1: Amino acid sequence(SEQ ID NO: 28) DYNMD Heavy chain FR2: Amino acid sequence(SEQ ID NO: 29) WVKQSHAKRLEWIG; Heavy chain CDR2: Amino acid sequence(SEQ ID NO: 30) DINPNYDSPTYSQKFKG; Heavy chain FR3: Amino acid sequence(SEQ ID NO: 31) RATLTVDNSSSTAYMELRSLTSEDTAVYYCARHeavy chain CDR3: Amino acid sequence (SEQ ID NO: 32) GHYYGYGYFDV;Heavy chain FR4: Amino acid sequence (SEQ ID NO: 33) WGAGTTVTVSS;Light chain: DNA sequence (381 bp) Signal sequence-FR1- CDR1 -FR2- CDR2-FR3- CDR3 -FR4 (SEQ ID NO: 10)ATGGAGTCACAGACTCAGGTCTTTGTATACATGTTGCTGTGGTTGTCTGGTGTTGATGGAGACAATGAGATGACCCAGTCTCAAAAATTCATGTCCACAGCAGTAGGAGACAGGGTCAGCGTCACC TGCAAGGCCAGTCAGAATGTGGGTACTAATGTAGCC TGGTATCAACAGAAACCAGGGAAATCTCCTAAAGTACTGATTTAC TCGGCATCCTACCGCTACAGT GGAGTCCCTGATCGCTTCAGAGGCAGTAGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACTTGGCAGAGTATTTCTGT CAGCAATATAACACCTATCCGTGGACG TTCGGTGGAGGCACCAAGCTGGAAATCAAA; Light chain FR1: DNA sequence (SEQ ID NO: 34)GACATTGAGATGACCCAGTCTCAAAAATTCATGTCCACAGCAGTAGGAGACAGGGTCAGC GTCACCTGC;Light chain CDR1: DNA sequence (SEQ ID NO: 35)AAGGCCAGTCAGAATGTGGGTACTAATGTAGCC Light chain FR2: DNA sequence(SEQ ID NO: 36) TGGTATCAACAGAAACCAGGGAAATCTCCTAAAGTACTGATTTAC;Light chain CDR2: DNA sequence (SEQ ID NO: 37) TCGGCATCCTACCGCTACAGT;Light chain FR3: DNA sequence (SEQ ID NO: 38)GGAGTCCCTGATCGCTTCAGAGGCAGTAGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACTTGGCAGAGTATTTCTGT; Light chain CDR3: DNA sequence(SEQ ID NO: 39) CAGCAATATAACACCTATCCGTGGACG;Light chain FR4: DNA sequence (SEQ ID NO: 40)TTCGGTGGAGGCACCAAGCTGGAAATCAAA;Light chain: Amino acid sequence (127 aa) Signal peptide-FR1- CDR1 -FR2-CDR2 -FR3- CDR3 -FR4 (SEQ ID NO: 11)MESQTQVFVYMLLWLSGVDGDIEMTQSQKFMSTAVGDRVSVTC KASQNVGTNVA WYQQKPG KSPKVLIYSASYRYS GVPDRFRGSRSGTDFTLTISNVQSEDLAEYFC QQYNTYPWT FGGGTKLE IK.Light chain FR1: Amino acid sequence (SEQ ID NO: 41)DIEMTQSQKFMSTAVGDRVSVTC; Light chain CDR1: Amino acid sequence(SEQ ID NO: 42) KASQNVGTNVA; Light chain FR2: Amino acid sequence(SEQ ID NO: 43) WYQQKPGKSPKVLIY; Light chain CDR2: Amino acid sequence(SEQ ID NO: 102) SASYRYS; Light chain FR3: Amino acid sequence(SEQ ID NO: 103) GVPDRFRGSRSGTDFTLTISNVQSEDLAEYFC;Light chain CDR3: Amino acid sequence (SEQ ID NO: 44) QQYNTYPWT;Light chain FR4: Amino acid sequence (SEQ ID NO: 45) FGGGTKLEIK;

Also provided is an anti-VCAM1 mAb V64-8 having the following sequences,where the Signal sequence/peptide is in italics, CDRs 1-3 areunderlined, and the framework (FR) regions are not italicized orunderlined.

Heavy chain: DNA sequence (396 bp) Signal sequence-FR1- CDR1 -FR2- CDR2-FR3- CDR3 -FR4 (SEQ ID NO: 12)ATGAACTTCGGGCTCAGCTTGATTTTCCTTGTCCCTATTTTAAAAGGTGTCCAGTGTGAAGTACAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGT AGTTATACCATGTCT TGGGTTCGCCAGTCTCCAGAGAAGAGGCTGGAGTGGGTCGCA GAGATTAGTAGTGGTGGTAGTTACACCCACTATGCAGCCAC TGTGACGGGCCGATTCACCATCTCCAGAGACAATGTCAAGAACACCCTGTACCTGGAAATGAGCAGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTGCAAGG GGAGAACTTTAC TGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA; Heavy chain FR1: DNA sequence(SEQ ID NO: 46)GAAGTACAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGT; Heavy chain CDR1: DNA sequence(SEQ ID NO: 47) AGTTATACCATGTCT; Heavy chain FR2: DNA sequence(SEQ ID NO: 48) TGGGTTCGCCAGTCTCCAGAGAAGAGGCTGGAGTGGGTCGCA;Heavy chain CDR2: DNA sequence (SEQ ID NO: 49)GAGATTAGTAGTGGTGGTAGTTACACCCACTATGCAGCCACTGTGACGGGC;Heavy chain FR3: DNA sequence (SEQ ID NO: 50)CGATTCACCATCTCCAGAGACAATGTCAAGAACACCCTGTACCTGGAAATGAGCAGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTGCAAGG; Heavy chain CDR3: DNA sequence(SEQ ID NO: 51) GGAGAACTTTAC; Heavy chain FR4: DNA sequence(SEQ ID NO: 52) TGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA;Heavy chain: Amino acid sequence (132 aa) Signal peptide-FR1- CDR1 -FR2-CDR2 -FR3- CDR3 -FR4 (SEQ ID NO: 13)MNFGLSLIFLVPILKGVQCEVQLVESGGGLVKPGGSLKLSCAASGFTFS SYTMS WVRQSPEKRLE WVAEISSGGSYTHYAATVTG RFTISRDNVKNTLYLEMSSLRSEDTAMYYCAR GELY WGQGT LVTVSA;Heavy chain FR1: Amino acid sequence (SEQ ID NO: 53)EVQLVESGGGLVKPGGSLKLSCAASGFTFS; Heavy chain CDR1: Amino acid sequence(SEQ ID NO: 54) SYTMS; Heavy chain FR2: Amino acid sequence(SEQ ID NO: 55) WVRQSPEKRLEWVA; Heavy chain CDR2: Amino acid sequence(SEQ ID NO: 56) EISSGGSYTHYAATVTG Heavy chain FR3: Amino acid sequence(SEQ ID NO: 57) RQSPEKRLEWVAEISSGGSYTHYAATVTG;Heavy chain CDR3: Amino acid sequence (SEQ ID NO: 58) GELY;Heavy chain FR4: Amino acid sequence (SEQ ID NO: 59) WGQGTLVTVSA;Light chain: DNA sequence (393 bp) Signal sequence-FR1- CDR1 -FR2- CDR2-FR3- CDR3 -FR4 (SEQ ID NO: 14)ATGAGTCCTGCCCAGTTCCTGTTTCTGTTAGTGCTCTGGATTCGGGAAACCAACGGTGATGTTGTGTTGACCCAGATTCCATCCACTTTGTCGGTTACCTTTGGACAACCAGCCTCCATCTCTTGC AAGGCAAGTCAGAGCCTCTTAGATAGAGGTGGAAAGACATTTTTCAAT TGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTAT CTGGTGTCTAAACTGGACTCT GGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTCTATTATTGC TGGCAAGGTACACATTTTCCGTGGACG TTCGGTGGAGGCACCAGACTGGAAATCAAA; Light chain FR1: DNA sequence(SEQ ID NO: 60)ATGTTGTGTTGACCCAGATTCCATCCACTTTGTCGGTTACCTTTGGACAACCAGCCTCCATC TCTTGC;Light chain CDR1: DNA sequence (SEQ ID NO: 61)AAGGCAAGTCAGAGCCTCTTAGATAGAGGTGGAAAGACATTTTTCAAT;Light chain FR2: DNA sequence (SEQ ID NO: 62)TGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTAT;Light chain CDR2: DNA sequence (SEQ ID NO: 63) CTGGTGTCTAAACTGGACTCT;Light chain FR3: DNA sequence (SEQ ID NO: 64)GGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTCTATTATTGC; Light chain CDR3: DNA sequence(SEQ ID NO: 65) TGGCAAGGTACACATTTTCCGTGGACG;Light chain FR4: DNA sequence (SEQ ID NO: 66)TTCGGTGGAGGCACCAGACTGGAAATCAAA;Light chain: Amino acid sequence (131 aa) Signal peptide-FR1- CDR1 -FR2-CDR2 -FR3- CDR3 -FR4 (SEQ ID NO: 15)MSPAQFLFLLVLWIRETNGDVVLTQIPSTLSVTFGQPASISC KASQSLLDRGGKTFFN WLLQRPGQSPKRLIY LVSKLDS GVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC WQGTHFPWT FGGGTRL EIK.Light chain FR1: Amino acid sequence (SEQ ID NO: 67)DVVLTQIPSTLSVTFGQPASISC; Light chain CDR1: Amino acid sequence(SEQ ID NO: 68) KASQSLLDRGGKTFFN; Light chain FR2: Amino acid sequence(SEQ ID NO: 69) WLLQRPGQSPKRLIY; Light chain CDR2: Amino acid sequence(SEQ ID NO: 70) LVSKLDS; Light chain FR3: Amino acid sequence(SEQ ID NO: 71) GVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC;Light chain CDR3: Amino acid sequence (SEQ ID NO: 72) WQGTHFPWT;Light chain FR4: Amino acid sequence (SEQ ID NO: 73) FGGGTRLEIK;

Also provided is an Anti-VCAM1 mAb V196-4 having the followingsequences, where the Signal sequence/peptide is in italics, CDRs 1-3 areunderlined, and the framework (FR) regions are not italicized orunderlined.

Heavy chain: DNA sequence (396 bp) Signal sequence-FR1- CDR1 -FR2- CDR2-FR3- CDR3 -FR4 (SEQ ID NO: 16)ATGAACTTCGGGCTCAGCTTGATTTTCCTTGTCCTTATTTTAAAAGGTGTCCATTGTGAAGTGCAGCTGGTGGAGTCTGGGGGAGCCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGTAGCCTCTGGATTCACTTTCAGT AGCTATGCCATGTCT TGGGTTCGCCAGTCTCCAGAGAAGAAGCTGGAGTGGGTCGCA GAAATTAGTAGTACTGGTAGTTACACCCACTATCCCGACACTG TGACGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGGAAATGAGCAGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTGCAAGA GGGGAGGCGCTC TGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA; Heavy chain FR1: DNA sequence(SEQ ID NO: 74)GAAGTGCAGCTGGTGGAGTCTGGGGGAGCCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGTAGCCTCTGGATTCACTTTCAGT; Heavy chain CDR1: DNA sequence(SEQ ID NO: 75) AGCTATGCCATGTCT; Heavy chain FR2: DNA sequence(SEQ ID NO: 76) TGGGTTCGCCAGTCTCCAGAGAAGAAGCTGGAGTGGGTCGCA;Heavy chain CDR2: DNA sequence (SEQ ID NO: 77)GAAATTAGTAGTACTGGTAGTTACACCCACTATCCCGACACTGTGACGGGC;Heavy chain FR3: DNA sequence (SEQ ID NO: 78)CGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGGAAATGAGCAGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTGCAAGA; Heavy chain CDR3: DNA sequence(SEQ ID NO: 79) GGGGAGGCGCTC; Heavy chain FR4: DNA sequence(SEQ ID NO: 80) TGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA;Heavy chain: Amino acid sequence (132 aa) Signal peptide-FR1- CDR1 -FR2-CDR2 -FR3- CDR3 -FR4 (SEQ ID NO: 17)MNFGLSLIFLVLILKGVHCEVQLVESGGALVKPGGSLKLSCVASGFTFS SYAMS WVRQSPEKKLE WVAEISSTGSYTHYPDTVTG RFTISRDNAKNTLYLEMSSLRSEDTAMYYCAR GEAL WGQGTS VTVSS;Heavy chain FR1: Amino acid sequence (SEQ ID NO: 81)EVQLVESGGALVKPGGSLKLSCVASGFTFS; Heavy chain CDR1: Amino acid sequence(SEQ ID NO: 82) SYAMS; Heavy chain FR2: Amino acid sequence(SEQ ID NO: 83) WVRQSPEKKLEWVA; Heavy chain CDR2: Amino acid sequence(SEQ ID NO: 84) EISSTGSYTHYPDTVTG; Heavy chain FR3: Amino acid sequence(SEQ ID NO: 85) RFTISRDNAKNTLYLEMSSLRSEDTAMYYCAR;Heavy chain CDR3: Amino acid sequence (SEQ ID NO: 86) GEAL;Heavy chain FR4: Amino acid sequence (SEQ ID NO: 87) WGQGTSVTVSS;Light chain: DNA sequence (393 bp) Signal sequence-FR1- CDR1 -FR2- CDR2-FR3- CDR3 -FR4 (SEQ ID NO: 18)ATGAGTCCTGCCCAGTTCCTGTTTCTGTTAGTGCTCTGGATTCGGGAAACCAACGGTGATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCGTTGGACAACCAGCCTCCATCTCTTGC AAGTCAAGTCATAGCCTCTTAGATAGTTATGGAAAGACATATTTGAAT TGGTTTTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTAT CTGGTGTCTAAACTGGACTC TGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGACTTTATTATTGC TGGCAGGGTACACATTTTCCGTGGACG TTCGGTGGAGGCACCAAGCTGGAAATCAAA; Light chain FR1: DNA sequence(SEQ ID NO: 88)GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCGTTGGACAACCAGCCTCCAT CTCTTGC;Light chain CDR1: DNA sequence (SEQ ID NO: 89)AAGTCAAGTCATAGCCTCTTAGATAGTTATGGAAAGACATATTTGAAT;Light chain FR2: DNA sequence (SEQ ID NO: 90)TGGTTTTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTAT;Light chain CDR2: DNA sequence (SEQ ID NO: 91) CTGGTGTCTAAACTGGACTC;Light chain FR3: DNA sequence (SEQ ID NO: 92)TGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGACTTTATTATTGC; Light chain CDR3: DNA sequence(SEQ ID NO: 93) TGGCAGGGTACACATTTTCCGTGGACG;Light chain FR4: DNA sequence (SEQ ID NO: 94)TTCGGTGGAGGCACCAAGCTGGAAATCAAA;Light chain: Amino acid sequence (131 aa) Signal peptide-FR1- CDR1 -FR2-CDR2 -FR3- CDR3 -FR4 (SEQ ID NO: 19)MSPAQFLFLLVLWIRETNGDVVMTQTPLTLSVTVGQPASISC KSSHSLLDSYGKTYLN WFLQRPGQSPKRLIY LVSKLDS GVPDRFTGSGSGTDFTLKISRVEAEDLGLYYC WQGTHFPWT FGGGTKLEIK; Light chain FR1: Amino acid sequence (SEQ ID NO: 95)DVVMTQTPLTLSVTVGQPASISC; Light chain CDR1: Amino acid sequence(SEQ ID NO: 96) KSSHSLLDSYGKTYLN; Light chain FR2: Amino acid sequence(SEQ ID NO: 97) WFLQRPGQSPKRLIY; Light chain CDR2: Amino acid sequence(SEQ ID NO: 98) LVSKLDS; Light chain FR3: Amino acid sequence(SEQ ID NO: 99) GVPDRFTGSGSGTDFTLKISRVEAEDLGLYYC;Light chain CDR3: Amino acid sequence (SEQ ID NO: 100) WQGTHFPWT;Light chain FR4: Amino acid sequence (SEQ ID NO: 101) FGGGTKLEIK.

In other embodiments, a monoclonal antibody to MAEA for use in thepresent methods, compositions, and kits may be a monoclonal antibody toMAEA available from R&D Systems (MAB7288), and a recombinant mousemonoclonal antibody to human MAEA is available from Creative Biolabs. Inother embodiments, a monoclonal antibody to VCAM1 for use in the presentmethods, compositions, and kits may be a monoclonal antibody availablefrom, e.g., Thermo Fisher Scientific, Abcam, Sigma-Aldrich, and Abnova.VCAM1 monoclonal antibodies are also described in US2010/0172902,incorporated herein by reference.

The antibody can be a human antibody or a humanized antibody or achimeric antibody. As used herein, a “human antibody” unless otherwiseindicated is one whose sequences correspond to (i.e., are identical insequence to) an antibody that could be produced by a human and/or hasbeen made using any of the techniques used for making human antibodies,but not one which has been made in a human. “Chimeric antibodies” areforms of non-human (e.g., murine) antibodies that contain humansequences in the constant domain regions of the antibody in order toeliminate or reduce immunogenic effects. “Humanized” forms of non-human(e.g., murine) antibodies are chimeric antibodies that also containhuman sequences in the variable domain regions of the antibody and thuscontain minimal sequence derived from non-human immunoglobulin. Ingeneral, a humanized antibody can comprise substantially all of at leastone, and typically two, variable domains, in which all or substantiallyall of the hypervariable loops correspond to those of a non-humanimmunoglobulin, and all or substantially all of the framework regionsare those of a human immunoglobulin sequence. In one embodiment, ahumanized antibody is a human immunoglobulin (recipient antibody) inwhich residues from a hypervariable region (HVR) of the recipient arereplaced by residues from a HVR of a non-human species (donor antibody)such as mouse, rat, rabbit, or nonhuman primate having the desiredspecificity, affinity, and/or capacity. For example, the antibody toMAEA could be a human or humanized antibody having the CDRs of MAB7288(which is a mouse IgG1 Ab). Techniques to humanize a monoclonal antibodyare well known and are described in, for example, U.S. Pat. Nos.4,816,567; 5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761;5,693,762; 5,585,089; and 6,180,370, the content of each of which ishereby incorporated by reference in its entirety.

Also provided is a monoclonal antibody to MAEA and a monoclonal antibodyto VCAM1. The term “monoclonal antibody” as used herein refers to anantibody member of a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible mutations, e.g., naturally occurring mutations, thatmay be present in minor amounts. Thus, the modifier “monoclonal”indicates the character of the antibody as not being a mixture ofdiscrete antibodies. In contrast to polyclonal antibody preparations,which typically include different antibodies directed against differentdeterminants (epitopes), each monoclonal antibody of a monoclonalantibody preparation is directed against a single determinant on anantigen. In addition to their specificity, monoclonal antibodypreparations are advantageous in that they are typically uncontaminatedby other immunoglobulins. Thus, an identified monoclonal antibody can beproduced by non-hybridoma techniques, e.g., by appropriate recombinantmeans once the sequence thereof is identified.

In certain embodiments, the antibody is a monoclonal antibody.Antibodies for use in the present methods, compositions, and kits mayinclude natural antibodies, synthetic antibodies, monoclonal antibodies,polyclonal antibodies, chimeric antibodies, humanized antibodies,multispecific antibodies, bispecific antibodies, dual-specificantibodies, anti-idiotypic antibodies, or fragments thereof that retainthe ability to bind a specific antigen, for example, VCAM1 or MAEA.

Compositions comprising the antibodies provided herein can additionallycomprise stabilizers to prevent loss of activity or structural integrityof the protein due to the effects of denaturation, oxidation, oraggregation over a period of time during storage and transportationprior to use. The compositions can comprise one or more of anycombination of salts, surfactants, pH and tonicity agents such as sugarscan contribute to overcoming aggregation problems. Where a compositionof the present disclosure is administered as an injection, it isdesirable to have a pH value in an approximately neutral pH range, it isalso advantageous to minimize surfactant levels to avoid bubbles in theformulation which are detrimental for injection into subjects. In anembodiment, the compositions can be in liquid form and stably supportshigh concentrations of bioactive antibody in solution and is suitablefor inhalational or parenteral administration. In an embodiment, thecomposition is suitable for intravenous, intramuscular, intraperitoneal,intradermal and/or subcutaneous injection. In an embodiment, thecomposition is in liquid form and has minimized risk of bubble formationand anaphylactoid side effects. In an embodiment, the composition isisotonic. In an embodiment, the composition has a pH or 6.8 to 7.4.

Compositions comprising the antibodies or fragments thereof providedherein can also be lyophilized or provided in any suitable formincluding, but not limited to, injectable solutions, inhalablesolutions, gel forms, or tablet forms.

Compositions comprising the antibodies or fragments thereof providedherein can be administered to the subject in a pharmaceuticalcomposition comprising the antibody or fragment and a pharmaceuticallyacceptable carrier. The term “carrier” is used in accordance with itsart-understood meaning, to refer to a material that is included in apharmaceutical composition but does not abrogate the biological activityof the antibody or antibody fragment included within the composition.Pharmaceutically acceptable carriers include, for example, sterileisotonic saline, phosphate buffered saline solution, water, andemulsions, such as an oil/water or water/oil emulsions.

The antibody or antibody fragment can be conjugated with a cytotoxicagent.

The antibody or antibody fragment can be administered to subjects usingroutes of administration known in the art, including, but are notlimited to, intravenous, intramuscular and intraperitonealadministration.

Also provided are a blocking antibody to VCAM1, an antibody fragmentthat blocks the activity of VCAM1, a blocking antibody to MAEA, and anantibody fragment that blocks the activity of MAEA for use as amedicament in treatment of cancer, in inhibiting engraftment of leukemiacells such as AML, CML, PV, ET, ALL, and CLL cells, and in enhancing theefficacy of cytarabine for treatment of cancer, wherein the antibody orantibody fragment is specific for VCAM1 or MAEA. The cancer can be, forexample, one or more of bladder, breast, brain, colorectal, kidney,oesophagus, gastrointestinal tract, liver, lung, ovarian, pancreas,prostate, skin, stomach, and uterine cancer, melanoma, myelodysplasticsyndrome (MDS) (a pre-leukemia), non-Hodgkin lymphoma, and a hematologicmalignancy. Hematologic malignancies can derive from myeloid or lymphoidcell lines. Lymphomas, lymphocytic leukemias, and myeloma are from thelymphoid line, while acute and chronic myelogenous leukemia,myelodysplastic syndromes and myeloproliferative diseases are myeloid inorigin. The hematologic malignancy can be a myeloproliferative disease.The hematologic malignancy can be, for example, AML, CML, PV, ET, ALL,or CLL.

The present disclosure provides a method of treating a condition (e.g.,a cancer or sickle cell disease) in a subject comprising administeringto the subject an antibody or antibody fragment in an amount effectiveto inhibit the activity of VCAM1 and/or an antibody or antibody fragmentin an amount effective to inhibit the activity MAEA to treat thecondition in a subject, wherein the antibody or antibody fragment isspecific for VCAM1 or MAEA.

As used herein, the term “treat” a cancer means to eradicate the cancerin a subject, or to reduce the size of a cancer or cancer tumor in thesubject, or to stabilize a cancer or cancer tumor in the subject so thatit does not increase in size, or to prevent or reduce the spread of thecancer in the subject.

As used herein, the term “treat” sickle cell disease (SCD) means toreduce the acute (e.g., vaso-occlusion) or chronic (e.g., organ damage)manifestations of SCD.

A “therapeutically effective amount” of a composition as used herein isan amount of a composition that produces a desired therapeutic effect ina subject, such as treating cancer, treating SCD, or treating PV. Incertain embodiments, the therapeutically effective amount is an amountof the composition that yields maximum therapeutic effect. In otherembodiments, the therapeutically effective amount yields a therapeuticeffect that is less than the maximum therapeutic effect. For example, atherapeutically effective amount may be an amount that produces atherapeutic effect but that also avoids one or more side effects that isassociated with a dosage that yields the maximum therapeutic effect. Atherapeutically effective amount for a particular composition will varybased on a variety of factors, including but not limited to thecharacteristics of the therapeutic composition (e.g., activity,pharmacokinetics, pharmacodynamics, and bioavailability), thephysiological condition of the subject (e.g., age, body weight, sex,disease type and stage, medical history, general physical condition,responsiveness to a given dosage, and other present medications), thenature of any pharmaceutically acceptable carriers in the composition,and the route of administration. One skilled in the clinical andpharmacological arts will be able to determine a therapeuticallyeffective amount through routine experimentation, namely by monitoring asubject's response to administration of a composition and adjusting thedosage accordingly. For additional guidance, see, e.g., Remington: TheScience and Practice of Pharmacy, 22nd Edition, Pharmaceutical Press,London, 2012, and Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 12th Edition, McGraw-Hill, New York, N.Y., 2011, theentire disclosures of which are incorporated by reference herein.

The cancer can be, for example, one or more of bladder, breast, brain,colorectal, kidney, oesophagus, gastrointestinal tract, liver, lung,ovarian, pancreas, prostate, skin, stomach, and uterine cancer,melanoma, non-Hodgkin lymphoma, myelodysplastic syndrome (MDS) (apre-leukemia), and a hematologic malignancy. Hematologic malignanciescan derive from myeloid or lymphoid cell lines. Lymphomas, lymphocyticleukemias, and myeloma are from the lymphoid line, while acute andchronic myelogenous leukemia, myelodysplastic syndromes andmyeloproliferative diseases are myeloid in origin. The hematologicmalignancy can be a myeloproliferative disease. The hematologicmalignancy can be, for example, acute myeloid leukemia (AML), chronicmyeloid leukemia (CML), polycythemia vera (PV), essential thrombocytosis(ET), acute lymphoblastic leukemia (ALL), or chronic lymphocyticleukemia (CLL).

The treatment can comprise administering to a subject a combination oftwo or more of:

-   -   a) a blocking antibody to VCAM1 or an antibody fragment that        blocks the activity of VCAM1, wherein the antibody or antibody        fragment is specific for VCAM1;    -   b) a blocking antibody to MAEA or an antibody fragment that        blocks the activity of MAEA, wherein the antibody or antibody        fragment is specific for MAEA;    -   c) one or more chemotherapeutic agents; and    -   d) one or more immune system enhancing agents;    -   wherein the combination includes at least a) or b).

The different components of the combination can be administered at thesame time, sequentially, or one spaced in time before the other. Incertain embodiments of the methods, compositions, and kits providedherein, an VCAM1 inhibitor or MAEA inhibitor, e.g., a VCAM1 or MAEAantibody or antibody fragment, may be administered together as part ofthe same composition. In other embodiments of the methods providedherein, the MAEA inhibitor and the VCAM1 inhibitor may both beadministered separately, i.e., as separate compositions. In theseembodiments, the inhibitors may be administered sequentially orsimultaneously, and may be administered via the same or differentroutes. In those embodiments where the inhibitors are administeredsequentially, they may be administered at the same or differentintervals. For example, one inhibitor may be administered morefrequently than the other or may be administered over a longer timecourse. In certain of these embodiments, one inhibitor may beadministered one or more times prior to the first administration of thesecond inhibitor. When administration of the second inhibitor isinitiated, administration of the first inhibitor may either cease orcontinue for all or part of the course of administration of the secondinhibitor. In certain embodiments wherein the MAEA inhibitor or theVCAM1 inhibitor is MAEA antagonist antibody or a VCAM1 antagonistantibody, the antibody may be administered two or more times per day,daily, two or more times per week, weekly, bi-weekly (i.e., every otherweek), every third week, or monthly. In certain embodiments, theantibody is administered weekly, bi-weekly, or every third week, ormonthly. In certain embodiments, the MAEA inhibitor and/or the VCAM1inhibitor may be administered for a specific time course determined inadvance. For example, the MAEA and/or VCAM1 inhibitors may beadministered for a time course of 1 day, 2 days, 3, days, 4 days, 5days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7weeks, 8 weeks, 9 weeks, or 10 weeks. In other embodiments, the MAEAand/or VCAM1 inhibitors may be administered indefinitely, or until aspecific therapeutic benchmark is reached. For example, the MAEA and/orVCAM1 inhibitors may be administered until tumor growth is arrested orreversed, until one or more tumors are eliminated, or until the numberof cancer cells are reduced to a specific level.

The one or more chemotherapeutic agents can be, for example, but notlimited to, cytarabine (cytosine arabinoside or ara-C), an anthracyclinedrug (such as, e.g., daunorubicin (daunomycin), idarubicin, and/ormitoxantrone), cladribine (2-CdA), fludarabine (Fludara®), topotecan,etoposide (VP-16), 6-thioguanine (6-TG), hydroxyurea (Hydrea®), acorticosteroid drug (such as, e.g., prednisone or dexamethasone(Decadron®)), methotrexate (MTX), 6-mercaptopurine (6-MP), azacitidine(Vidaza®), and/or decitabine (Dacogen®).

The one or more immune system enhancing agents can be, for example, butnot limited to, an inhibitor of CD47 (also called Cluster ofDifferentiation 47 and integrin associated protein (IAP)), PD-1 (alsocalled Programmed cell death protein 1)/PD-L1 (also called Programmeddeath-ligand 1, Cluster of Differentiation 274 (CD274) and B7 homolog 1(B7-H1)), CTLA-4 (also called cytotoxic T-lymphocyte-associated protein4 and CD152 (Cluster of Differentiation 152)), CD200 (also calledCluster of Differentiation 200 or OX-2 membrane glycoprotein)/CD200R(CD200 receptor), LAG-3 (also called Lymphocyte-activation gene 3protein), TIM-3 (also called T-cell immunoglobulin and mucin-domaincontaining-3), VISTA (also called V-domain Ig suppressor of T cellactivation), or TIGIT (also called T cell immunoreceptor with Ig andITIM domains). The agent that inhibits the activity of, for example,CD47 can be, for example, a blocking antibody to CD47 or an antibodyfragment that blocks the activity of CD47, where the antibody orantibody fragment is specific to CD47. Examples of blocking antibodiesto CD47 are described in US2016/0137733, US2016/0137734 andUS2017/0081407, hereby incorporated by reference. The agent thatinhibits the activity of CD47 can also be a construct having a SIRPalpha domain or variant thereof. Such constructs are described, forexample, in US2015/0071905, US2015/0329616, US2016/0177276,US2016/0186150, and US20170107270, hereby incorporated by reference.

Also provided is a method of inhibiting engraftment of leukemia cells ina subject, the method comprising administering to the subject anantibody or antibody fragment in an amount effective to inhibit theactivity of vascular cell adhesion molecule 1 (VCAM1) and/or an antibodyor antibody fragment in an amount effective to inhibit the activity ofmacrophage erythroblast attacher (MAEA) to inhibit leukemia cellengraftment in a subject, wherein the antibody or antibody fragment isspecific for VCAM1 or MAEA. The leukemia cells can be, for example,acute myeloid leukemia (AML), chronic myeloid leukemia (CML),polycythemia vera (PV), essential thrombocytosis (ET), acutelymphoblastic leukemia (ALL), or chronic lymphocytic leukemia (CLL)cells.

Still further provided is a method of enhancing the efficacy ofcytarabine for treating a cancer in a subject, comprising administeringto the subject an antibody or antibody fragment in an amount effectiveto inhibit the activity of VCAM1 and/or an antibody or antibody fragmentin an amount effective to inhibit the activity of MAEA in combinationwith cytarabine to enhance the efficacy of cytarabine for treating acancer in a subject, wherein the antibody or antibody fragment isspecific for VCAM1 or MAEA. The cancer can be, for example, one or moreof AML, CML, PV, ET, ALL, CLL or non-Hodgkin's lymphoma.

All combinations of the various elements described herein are within thescope of the invention unless otherwise indicated herein or otherwiseclearly contradicted by context.

From the foregoing, it will be appreciated that specific embodiments ofthe invention have been described herein for purposes of illustration,but that various modifications may be made without deviating from thescope of the invention. Accordingly, the invention is not limited exceptas by the appended claims.

The following examples are provided in order to better illustrate theclaimed invention and are not to be interpreted as limiting the scope ofthe invention. To the extent that specific materials are mentioned, theyare only mentioned for purposes of illustration and are not intended tolimit the invention. One skilled in the art may develop equivalent meansor reactants without the exercise of inventive capacity and withoutdeparting from the scope of the present invention. It will be understoodthat many variations can be made in the procedures described hereinwhile still remaining within the bounds of the present invention. Theinventors intend such variations to be included within the scope of theinvention. One skilled in the art will readily appreciate that thespecific methods and results discussed are merely illustrative of theinvention as described more fully in the claims that follow thereafter.

EXAMPLES Example 1: Anti-VCAM1 Therapies

VCAM1 is expressed on hematopoietic stem and progenitor cells (HSPCs,FIG. 1A). Although VCAM1 expression in endothelial cells and itsfunctional implications have been extensively described, the role ofVCAM1 on HSCs has not been explored. Recent studies also suggest thatVCAM1 expression on endothelial and bone marrow (BM) stromal cells maymediate in part leukemic cell resistance to conventional chemotherapy.VCAM1 is more highly expressed on acute myelogenous leukemia (AML) cellsthan their healthy counterparts (FIGS. 1B, 1C). Since Csf1r-iCre miceexhibit broad Cre expression in all hematopoietic cells, including mostHSCs (FIG. 2A) and deletion of VCAM1 gene is embryonically lethal, VCAM1floxed mice were bred with a Csf1r-iCre transgenic line (referred to asVCAM^(Δ/Δ)) to investigate VCAM1's function postnatally. In this modelVCAM1 was efficiently depleted in phagocytic cells and also HSCs (FIG.2B). VCAM1 deletion in Csf1r-icre+ cells induced HSPC mobilization intothe peripheral blood; however, it did not significantly impairhematopoiesis (FIGS. 2C-2G). VCAM1 deletion did not increase the numberof apoptotic HSCs as determined by Annexin V staining (FIG. 3A) and nosignificant changes were observed in the proportion of cycling HSCs orgenes involved in HSC quiescence/proliferation (FIGS. 3B-3D).VCAM1^(Δ/Δ) and control mice challenged with 5-FU did not reveal anydeficit in hematopoietic stem and progenitor recovery (FIG. 3E).

To test whether VCAM1 antibody blockade can improve conventionalchemotherapy in animals with established disease, AML was established inimmunocompetent C57BL/6 recipients and then therapy of moribund leukemicmice was initiated with a daily injection of IgG control, anti-VCAM1,cytarabine, or a combination of anti-VCAM1/cytarabine. Anti-VCAM1antibody inhibition synergised with conventional chemotherapy to clearleukemic stem cells (LSCs) while sparing healthy HSCs, significantlyprolonging mice survival (FIG. 4 ). The viability of targeting VCAM1 asa therapeutic strategy was investigated by injecting healthy wild-typemice with anti-VCAM1 antibody. After treatment, mice appeared healthyand body, liver and spleen weighs were unaltered (FIGS. 5A-5D). Completeblood counts showed no hematopoietic defects but did indicate a smallincrease in the percentage of reticulocytes (FIG. 5E). These resultsindicate that targeting VCAM1 function with a blocking monoclonalantibody should be well tolerated and a promising therapeutic strategy.Analysis of The Cancer Genome Atlas (TCGA) databases indicated that highVCAM1 expression was associated with poor prognosis in human AMLpatients (FIGS. 6A, 6B). Furthermore, anti-VCAM1 treatment was able tosignificantly extend the survival of immunocompromised mice transplantedwith human primary AML samples (FIG. 6C).

Analysis of a recently published RNA-sequencing dataset of 675 humancancer cell lines indicated that >50% of those lines express VCAM1 (FIG.7A). In fact, different tissue types of human cancer cell lines expresshigh levels of VCAM1, in particular kidney, colorectal and pancreas(FIG. 7B),⁴⁵ and significant association of VCAM1 gene alterations werefound with many human cancer types (FIG. 8 ).

These studies demonstrate that VCAM1 is upregulated on malignanthematopoietic cells and that inhibition of binding of VCAM1 to itsreceptors will promote cancer cell clearance. These studies alsoindicate that this cell clearance mechanism is likely via a“don't-eat-me” signal since incubation of VCAM1^(Δ/Δ) AML cells withmacrophages led to enhanced phagocytosis of leukemic cells. This effectdid not result from a reduced expression of CD47, since CD47 expressionwas not altered in VCAM1^(Δ/Δ) mice. Monoclonal antibodies either aloneor in combination with treatment such as cytarabine are an effectivetreatment for cancer.

Example 2: VCAM1 Confers Innate Immune Tolerance on Hematopoietic andLeukemic Stem Cells

Haematopoietic stem cells (HSCs) home to the bone marrow (BM) via, inpart, the interactions with vascular cell adhesion molecule-1(VCAM1).⁵⁷⁻⁵⁹ Upon migrating into the BM, HSCs are vetted byperivascular phagocytes to ensure their self-integrity. In this Example,results show that VCAM1 is also expressed on healthy HSCs andupregulated on leukemic stem cells (LSCs) where it serves as aquality-control checkpoint for entry into BM by providing ‘don't-eat-me’stamping in the context of major histocompatibility complex (MHC)class-I presentation. While MHC haplotype-mismatched HSCs can engraftthe BM of recipient mice, conditional VCAM1 deletion, in the setting ofhaplotype mismatch, leads to impaired hematopoietic recovery due to HSCrecognition and clearance by phagocytes. Mechanistically, MHC mismatchedHSCs are recognized at least in part by paired Ig-like receptor-B(PIR-B) expressed on murine phagocytic myeloid cells. VCAM1 is also usedby cancer cells to escape immune detection as its expression isupregulated in multiple cancers, including acute myeloid leukemia (AML),where high expression is significantly associated with poor prognosis.In AML mouse models, VCAM1 promotes disease progression while VCAM1inhibition or deletion significantly reduces the leukemia burden andextends the survival of mice. These results suggest that VCAM1engagement regulates a critical immune checkpoint gate in the BM andoffers a novel strategy to eliminate cancer cells via modulation of theinnate immune tolerance.

HSCs possess the ability to replenish the haematopoietic systemfollowing transplantation into marrow-ablated recipients.⁶⁰ VCAM1, avascular endothelial and stromal adhesion molecule, is required forvascular development⁶¹ and known to mediate the entry^(62,63) andegress⁶³⁻⁶⁵ of HSCs and progenitors between BM and the bloodcirculation. Using flow cytometry analysis, Applicants found that VCAM1was also expressed at high levels on the majority of HSCs (˜75%) andsome progenitors in the BM, and its expression was downregulated in HSCsmobilized in the periphery (spleen and blood; FIGS. 1A, 13A-13D). Micecarrying floxed VCAM1 alleles (VCAM^(fl/fl))⁶⁶ were bred with aCsf1r-iCre transgenic line⁶⁷ (hereafter referred to asVCAM1^(Csf1r-iCre)) to evaluate its role on macrophages oferythroblastic islands.⁶⁸ Applicants have also found, however, high Credeletion efficiency in HSCs and their descendants consistent with thereported Csf1r expression in HSCs⁶⁹⁻⁷⁰ (FIGS. 2B, 13E). To evaluateVCAM1's function on HSCs, VCAM1^(Csf1r-iCre) BM cells were transplantedin lethally irradiated wild-type C57BL/6 recipients (FIG. 14A).Interestingly, the vast majority (˜75%) of recipients transplanted withVCAM1^(Csf1r-iCre) BM cells succumbed between days 14-28 (FIG. 14B). Norepopulating contribution from VCAM1Csf1r-iCre was also obtained incompetitive reconstitution experiments (FIGS. 14B, 14D). By contrast,the constitutive deletion of VCAM1 (without transplantation) did notalter HSC numbers, location, cycling status, or the numbers ofmultipotent progenitors (MPPs), colony-forming progenitors or blood cellcounts (FIGS. 2C-2E, 2G, 3A, 3B, 3E). In addition, 5-FU treatment didnot reveal any deficit in VCAM^(Csf1r-iCre) HSPC recovery (FIG. 3E).However, VCAM1 deletion led to reduced numbers of splenic HSCs andprogenitors (FIG. 2G) with slight augmentation of circulatingprogenitors in blood (FIG. 2F), in line with a previous report.⁷¹ Theseresults suggest that VCAM1 expressed on HSCs may regulate theirengraftment in the BM, but may be dispensable for steady-state or stresshematopoiesis.

To resolve the discrepancy between the dramatic HSC transplantationphenotype and the absence of constitutive HSC phenotype, the fate ofinjected VCAM1^(Csf1r-iCre) and VCAM1^(fl/fl) BM cells was tracked aftertransplantation. While no difference in homing to BM 3 h after injectionin lethally irradiated recipients was found (FIG. 16A), a time-coursefollow up revealed that the contribution of VCAM1^(Csf1r-iCre)progenitors to the recipient blood was reduced by ˜35% on day 4 and wasbarely detectable two weeks after transplantation (FIG. 9B).Post-transplantation failure was due to clearance by phagocytic cells asCFDA-SE fluorescently labelled VCAM1^(Csf1r-iCre) Lineage⁻ cellsaccumulated in host immune phagocytes (Gr-1high and Gr-1low monocytes,macrophages and neutrophils) after injection (FIGS. 9C, 9D, 16B). Thiswas accompanied by increased numbers of host phagocytic monocytes andneutrophils in VCAM1^(Csf1r-iCre)-transplanted recipient mice comparedto those transplanted with VCAM1^(fl/fl) BM cells (FIG. 10C). Toascertain that the clearance was not dependent on the damaged inflictedby irradiation, applicant assessed the BM chimerism in G-CSF-mobilizedparabiotic mice in which animals share the blood circulation. Theseresults showed that while VCAM1^(fl/fl) HSCs and MPPs significantlyengrafted the partner mice, VCAM1^(Csf1r-iCre) cells did not engraft theparabiont partner (FIGS. 9E, 9F). These data indicate thatVCAM1-deficient HSCs are susceptible to be recognized by host phagocyticcells in the BM.

In the course of studies to identify the immunological mechanismunderlying the engraftment failure of VCAM1-deficient HSCs,VCAM1^(Csf1r-iCre) mice—where Csf1r-iCre transgenic mice generated inthe FVB background were backcrossed over 10 generations into the C57BL/6background-remained heterozygote for the MHC haplotypes H-2q (from FVB)and H-2b (from C57BL/6) (FIG. 17A). Indeed, the Csf1r-iCre transgene wasgenetically linked to the MHC locus with a frequency of meioticrecombination estimated at 7.87% (FIG. 17B). Accordingly,VCAM^(Csf1r-iCre) and VCAM1^(fl/fl) control mice were bred to generateeither syngeneic (H-2^(b/b)) or haplotype mismatched (H-2^(b/q)) MHCstatus and to evaluate VCAM1's function on HSC engraftment in thecontext of syngeneic or haplotype-mismatched transplantation (FIG. 10A).These results show that VCAM1 deletion led to striking defects inhematopoietic engraftment only in the context of MHChaplotype-mismatched transplantation (donor H-2^(b/q); recipientH-2^(b/b); FIGS. 10A-10D, 17C). Whereas donor BM cells carrying theVCAM1^(fl/fl) with haplotype-mismatched genotype exhibited engraftmentand survival similar to syngeneic counterparts, those VCAM1-deficientwith haplotype mismatch did not engraft and ˜76% of the animalstransplanted with these cells died (FIGS. 10A, 10B). Phagocytosis ofdonor HSCs in the BM was only triggered when HSCs were lacking VCAM1combined with the haplotype-mismatched genotype (FIG. 9C), as syngeneicVCAM1^(Csf1r-iCre);H-2^(b/b) cells were not targeted (˜0% phagocytosis,data not shown). As CD8⁺ cells are classical responders to MHC-Imismatch,⁷² Applicants evaluated their requirement for clearing theVCAM1^(Csf1r-iCre);H-2^(b/q) HSCs in vivo by antibody depletion of CD8⁺cells before transplantation. Results revealed that the survival defectof VCAM1^(Csf1r-iCre);H-2^(b/q) recipients could not be rescued by CD8⁺T cells depletion, suggesting that CD8⁺ cells were dispensable (FIGS.18A, 18B). Taken together, these results indicate that VCAM1 expressionon HSCs is required for vetting by phagocytes to provide entry in the BMmicroenvironment.

VCAM1 interacts with α4β1 integrin (also known as VLA-4), which isexpressed on most hematopoietic cells.⁷³ The mouse PIR-B receptor,expressed by myeloid phagocytes and B cells⁷⁴ provides negativeregulation of immune cells upon recognition of MHC-I molecules.⁷⁵Accordingly, PIR-B may cooperate in the anti-phagocytic activity ofVCAM1 and MHC-I. Flow cytometry analysis of PIR-B expression in BMimmune cells shows that the vast majority (>63%) of Gr1high and Gr1lowmonocytes express both PIR-B and the VCAM1 counterreceptor VLA4 whereasthese are expressed in much lower fractions in other immune cells (FIG.10E). The intracellular transduction of PIR-B inhibitory signalingcorrelates with the tyrosine phosphorylation (P-TYR) status of itsimmunoreceptor tyrosine-based inhibitory motifs.^(74,75) Accordingly, onday 6 after transplantation of VCAM^(Csf1ri-Cre);H-2^(b/q) cells, theP-TYR levels in host CD45.1;H-2^(b/b) PIR-B⁺ Gr1^(high) and Gr1^(low)monocytes and neutrophils were significantly reduced compared toVCAM1^(fl/fl);H-2^(b/q) control (FIGS. 10F, 10G). To evaluate furtherthe role of PIR-B, syngeneic and haplotype-mismatched VCAM^(Csf1r-iCre)and VCAM1^(fl/fl) BM cells were transplanted into Pirb^(−/−) mice⁷⁶ anddonor cell engraftment was evaluated by real-time PCR. In the absence ofPIR-B inhibitory signal, VCAM1 deletion led to significant reductions(˜60%) in the early (1 week) engraftment of syngeneicVCAM1^(Csf1r-iCre);H-2^(b/b) cells compared to VCAM^(fl/fl);H-2^(b/b)cells (FIG. 10H), suggesting that the absence of VCAM1 also promotescell clearance by syngeneic Pirb^(−/−) phagocytes. However, theengraftment levels in 5 out of 6 Pirb^(−/−) recipients of syngeneicVCAM1-null cells recovered to the levels of syngeneic VCAM1-sufficientcells, 4 weeks post-transplantation (FIG. 18C). As expected, due to thehyper-responsiveness of Pirb^(−/−) immune cells,⁷⁵ bothhaplotype-mismatched cohorts failed to engraft (FIG. 18C). These resultssuggest that PIR-B expression on phagocytes contributes to suppress theimmune response triggered by VCAM1-deficient HSCs and progenitors,although the transient phenotype suggests that other molecules may alsobe at play. That VCAM1 expression would provide innate immune toleranceindicated that this pathway may be of use for cancer cells. Upregulationin VCAM1 expression has been reported in various cancer cell types,including gastric,⁷⁷ renal,⁷⁸ hepatocellular,⁷⁹ acute promyelocyticleukaemia,⁸⁰ and breast cancer, where VCAM1 promotes metastasis to thelungs and bones by providing survival signals⁸¹ and recruitingosteolytic cells.⁸² Recent studies suggest that VCAM1 expression onendothelial and BM stromal cells may mediate in part leukemia cellresistance to conventional chemotherapy.⁸³⁻⁸⁶ Results provided hereinsuggest that cell autonomous expression of VCAM1 may confer immuneevasion. In an immunocompetent mouse model of AML driven by MLLAF931(FIG. 19A), results indicated that VCAM1 expression on AML cells was7-fold higher than BM hematopoietic cells, and that expression on LSCswas 4-fold higher than healthy HSCs (FIG. 1C).

Leukemic cells can upregulate CD47^(88,89) or MHC class-I⁸⁹ molecules toavoid phagocytosis or aberrantly express pro-phagocytic signalsincluding AML-specific neo-antigens and AML-associated antigens that canelicit anti-leukemia immune responses if the balance betweenanti-phagocytic and phagocytic signals is perturbed.⁷² To test theeffect of genetic VCAM1 deletion and MHC-mismatch on AML progression,VCAM1^(Csf1r-iCre);H-2^(b)/q and VCAM1^(fl/fl);H-2^(b/q) Lineage⁻ Sca-1⁺c-Kit⁺ (LSK) cells were transduced with the pMSCV-MLL-AF9-GFP oncogene.Strikingly, FACS analysis of primary AML recipient mouse BM revealed amarked reduction (>99%) of phenotypic VCAM1^(Csf1r-iCre) LSCs comparedto VCAM1^(fl/fl) control (FIGS. 11B, 19B). Moreover, whole-mountconfocal imaging of the sternal marrow showed little leukemiainfiltration of VCAM1^(Csf1r-iCre) AML compared to VCAM1^(fl/fl) AML(FIG. 1C). These results were further confirmed in aluciferase-expressing MLL-AF9 reporter line that allowed monitor tumorprogression using bioluminescence in live mice (FIG. 11D). Accordingly,the survival of secondary recipient mice receiving 20,000 sorted GFP⁺leukaemic cells from VCAM1^(fl/fl) or VCAM1^(Csf1r-iCre) primaryrecipients was significantly prolonged in mice harboringVCAM1^(Csf1r-iCre) AML cells relative to VCAM1^(fl/fl) AML (FIG. 11E).Of note, FACS analysis of the BM from moribund VCAM1^(Csf1r-iCre) AMLmice, revealed that >85% of LSCs in these mice expressed VCAM1 by day103 post-transplantation (FIG. 19C), suggesting that incomplete Crerecombination in Csf1r-iCre model may have allowed rare VCAM1⁺ LSCs toescape and colonize the marrow of secondary recipients, leading to theirdeath. These results suggest that ablation of VCAM1 significantlyimpairs AML engraftment and disease progression in vivo.

To investigate the requirements of phagocytic cells in the in vivoclearance of MHC mismatched VCAM1^(Csf1r-iCre) AML, phagocytes weredepleted by injection of clodronate liposomes (or control PBS liposomes)prior to and after transplant of VCAM1^(fl/fl) and VCAM1^(Csf1r-iCre)AML cells (FIGS. 11F, 19D). Engraftment was markedly enhanced byphagocyte depletion, and remarkably, the engraftment defect ofVCAM1^(Csf1r-iCre) AML cells was rescued (FIG. 11G), indicating animportant role for phagocytes in the establishment of VCAM1-deficientAML. To evaluate whether phagocytosis was preceded by the induction ofapoptosis, AML cells were incubated with IgG, anti-VCAM1 blockingantibody or camptothecin (a known inducer of AML apoptosis). Resultsshowed no difference in the percentage of apoptotic LSCs cells betweencontrol and anti-VCAM1 treated groups 4.5 hours after treatment (FIG.19E). Thus, these results suggest that clodronate-sensitive phagocytesplay a key role in AML clearance and that the phagocyte recognition doesnot require AML cell apoptosis. To evaluate the safety and efficacy ofVCAM1 inhibition in a clinical scenario in which the therapeuticintervention occurs after the disease is established, C57BL/6 syngeneicAML were allowed to develop (>50% circulating AML-GFP⁺ cells) inimmunocompetent C57BL/6 recipients, and then initiated therapy of thesemoribund leukemic mice with a daily injection of IgG1 control,anti-VCAM1, cytarabine (Ara-C), or a combination of anti-VCAM1/Ara-C for5 days (FIG. 4A). Remarkably, this very short treatment with anti-VCAM1antibody preferentially and significantly reduced the frequency andabsolute number of phenotypic BM LSCs in vivo (FIG. 4B). Next, a similartreatment strategy was used in mice harboring ˜20% circulating AML-GFP⁺cells. While the disease progressed in all IgG1-treated AML mice, it wasstabilized by anti-VCAM1 treatment and dramatically reduced in micetreated with anti-VCAM1 in combination with Ara-C (FIG. 4C),significantly extending the survival of the animals (FIG. 4D).Anti-VCAM1 treatment regimen resulted in 100% antibody coating of VCAM1⁺HSCs and appeared to be safe for healthy HSCs since it did not depletetheir numbers or altered blood counts (FIG. 4E).

As high VCAM1 is associated with reduced survival of patients with AMLin the TCGA database (FIG. 20A), the functional significance of elevatedVCAM1 in human AML cells was investigated. To this end, VCAM1 wasoverexpressed in MOLM-13 cells, which constitutively do not expressVCAM1 (FIGS. 20B, 20C), and transplanted hVCAM1-ZsGreen-transduced andZsGreen control (Mock)-transduced MOLM-13 cells into immunocompromisedNOD-scid Il2rg^(−/−) (NSG) mice. Results showed that VCAM1-expressingMOLM-13 exhibited rapid disease progression and significantly highernumbers of leukemic cells in peripheral blood compared to controlMOLM-13 (FIG. 20D), leading to a significant reduction in the survivalof hVCAM1-MOLM-13-transplanted mice (FIG. 20E). There was no differencein homing capacity of hVCAM1-MOLM-13 cells, in vivo cell viability orcycling (FIGS. 20F-20I). Importantly, anti-human VCAM1 administrationsignificantly extended the survival of the animals transplanted withhVCAM1-MOLM-13 cells (FIG. 20J). Thus, VCAM1 overexpression increasesthe tumorigenicity of human AML cells.

Next, VCAM1 expression was assessed in highly purified primary human AMLstem and progenitor cells relative to healthy age-matched controlsamples from published data of AML patients with normal karyotype,complex karyotype, and deletion of chromosome 7.^(90,91) Results fromthese experiments demonstrated that VCAM1 was significantlyoverexpressed in short-term repopulating HSCs (ST-HSCs), the compartmentmost enriched in functional human LSCs, particularly in the group withnormal AML karyotype (FIG. 20K). To assess the relevance of VCAM1 signalinhibition in human AML, primary human AML samples were transplantedinto NSG mice. Upon disease establishment (4-5 weeks post-transplant),the animals were treated with 2 courses of anti-human VCAM1 monoclonalantibody or control IgG1. Results demonstrated that VCAM1 inhibitionsignificantly prolonged in the survival of leukemic mice (FIGS. 12A,12B). Taken together, these results suggest that VCAM1 blockade is safein pre-clinical models to reduce the leukemic burden in vivo and maysynergize with Ara-C treatment to clear LSCs while sparing healthy HSCs.

The therapeutic outcomes in AML remain poor, with relapses representingthe major cause of treatment failure due to resistant disease. Theimmune system has emerged as a critical defense for preventing tumorinitiation and controlling tumor growth.^(88,89,90,92,93) Resultsprovided herein reveal a novel function for the HSC niche molecule VCAM1on hematopoietic and leukemic stem cells by acting cell-autonomously asa “don't-eat-me” signal in the context of MHC-I presentation. VCAM1regulates a critical vetting process by resident phagocytes in the BM toallow the entry of healthy or malignant stem cells. This vettingrequires parallel checkpoints by VCAM1 and MHC-I on the stem cells andtheir counter-receptors on phagocytes, where the absence or blockade ofVCAM1 combined with MHC mismatch instruct phagocytes to kill (FIG. 21C).In the context of a leukemia, VCAM1 inhibition appears sufficient togive the green light to kill tumor cells, even in the setting of animmunocompetent syngeneic host, likely due to the presence of tumorneoantigens which may be perceived by host phagocytes as non-self.Interestingly, as discussed below in greater detail in Example 5,results disclosed herein identify another erythroblastic island adhesionmolecule, the Macrophage-Erythroblast Attacher (MAEA), as required forAML development and progression. These studies suggest that anti-VCAM1therapy may synergize with conventional chemotherapy and may provideeffective combination treatments to enhance the innate immunity responseto cancer.

Example 3: Anti-VCAM1 Therapies

The VCAM-1 protein mediates the adhesion of lymphocytes, monocytes,neutrophils, eosinophils, basophils, and sickle red blood cells (RBCs)to the vascular endothelium.¹²⁷ VCAM1 is inducible by inflammatorycytokines such as TNF-alpha and ILL. VCAM-1 also functions inleukocyte-endothelial cell signal transduction and may play a role inthe development of atherosclerosis and rheumatoid arthritis (RA).¹²⁸Given VCAM1's role in leukocyte adhesion during inflammation, wereasoned that it may be an important target to protect SCD mice fromacute vaso-occlusion.

To test the effect of an anti-VCAM1 antibody on a murine model for SCD,mice were intravenously injected with 200 μg/mice rat IgG1 or anti-mouseVCAM-1 antibody (clone M/K-2.7 from BioxCell) at 16 hours and 2 hoursbefore TNF-α challenge (n=5, rat IgG1 antibody 200 μg/mice or VCAM1antibody 200 μg/mice). These results show that white blood cell (WBC)rolling was significantly increased (p<0.0001) in mice injected with ananti-VCAM1 antibody as compared to mice injected with rat IgG (FIG.39B). Additionally, the number of adhesions per 100 μm (FIG. 39C;p<0.0001) and interactions between RBCs and WBCs per minute (FIG. 39D;p<0.05) were significantly decreased in mice injected with an anti-VCAM1antibody as compared to mice injected with IgG. mice injected with ratIgG1 or VCAM1 antibody (no significant difference). FIG. 39E depictsextravasated WBCs in mice injected with rat IgG1 or VCAM1 antibody.Results further demonstrate that mice injected with an anti-VCAM1antibody have better survival than those injected with IgG (FIG. 39F).Treatment with an anti-VCAM1 antibody additionally resulted in asignificantly increased centerline velocity and lower shear rate ascompared mice injected with IgG (FIG. 39G). Collectively, these resultsindicate that blocking VCAM1 function may help treat SCD by reducing thenumber of adhesions, reducing WBC activation and RBC/WBC interactions,leading to increased centerline velocity, increased shear rate, andprolonged survival during the stress of vaso-occlusion.

Blood was harvested and total and differential counts were obtainedusing an Advia cell counter. FIGS. 40A-40D. FIGS. 40A-40D depictexperimental results quantifying white blood cell numbers and types inmice injected with rat IgG1 or VCAM1 antibody. FIG. 40A depicts a chartquantifying WBCs in mice injected with rat IgG1 or VCAM1 antibody(p<0.05 between the 2 groups). FIG. 40B depicts a chart quantifying theblood differential count (by percent) in mice injected with rat IgG1 orVCAM1 antibody (eosinophils=white; lymphocytes=grey; neutrophils=black).FIG. 40C depicts a chart quantifying the blood cell count in miceinjected with rat IgG1 or VCAM1 antibody (eosinophils=white;lymphocytes=grey; neutrophils=black). FIG. 40D is a chart depictingserum inflammatory cytokine levels (pg/ml) in mice injected with ratIgG1 or VCAM1 antibody.

Inventors next investigated if the VCAM1 receptor, the integrinalpha4beta1 (CD49d) was expressed on the surface of neutrophils by flowcytometry. FIG. 41 depicts a chart showing data indicating that theVCAM1 receptor CD49d is expressed on mouse neutrophils from SA and SSSCD mice.

Example 4: Anti-MAEA Therapies

Conditional MAEA knockout (MAEA^(floxed)) mice were generated andmacrophage MAEA expression deleted by Csf1r-Cre (FIGS. 21A, 21B, 22A,22B). Macrophage MAEA expression was determined to be required for BMmacrophage development and erythropoiesis at steady state (FIGS.21D-21F, 22C-22H). Based on a previous study that depletion ofmacrophages could normalize polycythemia vera, Applicants investigatedtreatment with anti-MAEA antibody would achieve similar effects.

Unexpectedly, MAEA^(Csf1r-Cre) mice also exhibited marked reductions incirculating leukocytes (FIG. 30D), due to a loss of B and T lymphocytes(FIG. 30D). This is likely due to MAEA expression on bone marrowhematopoietic stem and progenitor cells (HSPCs) (FIGS. 30A, 30B) and itsinvolvement in lymphoid commitment from the HSPCs (FIGS. 30E-30G).Importantly, MEAE expression was also required for successful HSCengraftment after bone marrow transplantation (FIG. 23A), and this isnot due to any microenvironmental defects (FIGS. 23B-23H). Without MAEA,HSCs are more actively cycling but do not show increased apoptosis(FIGS. 24A-24B). In addition, MAEA-deficient HSCs regenerate (FIGS. 24C,24D), home to BM (FIG. 24E), and form colonies (FIG. 24F) comparably tocontrol counterparts.

It was hypothesized that leukemia cells might hijack the same mechanismfor their progression. Indeed, significant association of MAEAamplification mutations was found with many human cancer types (FIG.25A), and MAEA up-regulation strongly correlated with poor prognosis inhuman AML patients (FIGS. 25B, 25D). MAEA expression is alsoup-regulated in a murine model of acute myeloid leukemia (FIGS. 26A,26B). By genetically deleting MAEA expression from the AML cells usingCsf1r-Cre and Mx1-Cre, MAEA expression was shown to be required for AMLengraftment and progression in vivo (FIGS. 26C-26I). Importantly,treating AML-bearing mice with a polyclonal anti-MAEA antibodysignificantly reduced their circulating leukemia cells (FIG. 26J), butdid not cause overt toxicity in healthy mice (FIG. 27 ). Lastly,analysis of human cancer cell lines revealed a broad expression of MAEAacross cancer types (FIG. 19 ).

These results indicate that MAEA is a novel adverse prognosis factor anddrug target expressed on malignant hematopoietic and other cancer cells,and that MAEA is a target to promote cancer cell clearance by the hostimmune system.

Example 5: Anti-MAEA Antibodies. MAEA Expressed by Macrophages, but notErythroblasts, Maintains Postnatal Bone Marrow Erythroblastic Islands

Red blood cell (RBC) homeostasis is tightly regulated by balancedproduction and clearance. Bone marrow (BM) erythroid precursors werefirst observed several decades ago in tight association with a centralmacrophage in a structure referred to as erythroblastic island (EI).¹Macrophages regulate both normal and diseased erythropoiesis, includingpromotion of erythroid precursor survival and proliferation, ironhomeostasis and transfer, and terminal maturation and enucleation.²⁻⁵These activities are promoted by direct interactions between themacrophages and erythroblasts^(6,7) via several proposed adhesionmechanisms including (macrophage: erythroblast) VCAM1: VLA-4,^(8,9) αV:Icam4,¹⁰ or MAEA: MAEA,⁷ CD163,¹¹ and Palladin¹². However, the exactrole of these adhesion molecules during in vivo adult erythropoiesis hasnot been determined.

Among these, MAEA was originally identified as an adhesion moleculeexpressed by both macrophages and erythroblasts and suggested to mediateEI formation via its homophilic interactions.^(7,13) Targeted geneinactivation of MAEA caused severe defects in fetal liver erythropoiesisand macrophage development,¹⁴ but the perinatal lethality of MAEA-nullembryos has prevented detailed examination of its function in adulthematopoiesis. In this study, a conditional allele of MAEA wasgenerated. MAEA was determined to play a critical role in adult BMmacrophage development and EI function. Comparative analysis with VCAM1deletion shows that MAEA exerts a dominant role in the EI. Selectivedeletion of MAEA in macrophage or erythroblast shows only disruption ofBM erythropoiesis when MAEA is deleted in macrophage, suggesting thatMAEA may not interact by homophilic interactions.

Methods

Animals. MAEA^(fl/fl) mice were generated as described below.VCAM1^(fl/fl) mice¹⁵ were kindly provided by Dr. Thalia Papayannopoulouand backcrossed to C57BL/6 strain for at least 10 generations. Csf1r-Cremice¹⁶ were a gift from Dr. Jeffrey W. Pollard (University of Edinburgh)and also backcrossed onto C57BL/6 background. CD169-Cre knockin micewere previously generated and described¹⁷. Epor-Cre mice¹⁸ were kindlyprovided by Dr. Ann Mullally (Dana-Farber/Brigham and Women's Hospital).C57BL/6 (CD45.2) and B16-Ly5.1 (CD45.1) mice were purchased from CharlesRiver Laboratories (Frederick Cancer Research Center, Frederick,Md.)/NCI or the Jackson Laboratories (B6.SJL-Ptprc^(a) Pepc^(b)/BoyJ).R26-tdTomato (B6.Cg-Gt(ROSA)26Sor^(tm14(CAG-tdTomato)Hze)/J) and Mx1-Cre(B6.Cg-Tg(Mx1-cre)1Cgn/J) mice were obtained from Jackson Laboratories.All animals were housed in specific pathogen-free barrier facility. Allexperimental procedures were approved by the Animal Care and UseCommittee of Albert Einstein College of Medicine. All experiments wereperformed on mice of both genders with littermate controls from the samecolony between 6-12 weeks of age.

Generation of MAEA^(fl/fl) mice. The EuMMCR targeting vectorPG00141_Z_1_G10 was purchased and electroporated into WW6 embryonic stem(ES) cells. After drug selection, resistant ES cell colonies were pickedand screened by Southern blot analysis using 5′ and 3′ external probes.Correctly targeted ES cell clones were injected into C57BL/6 blastocystsand the resulting chimeric mice were bred with C57BL/6 animals toestablish the MAEA^(targeted) line. Once the germline transmission wasconfirmed, the MAEA^(targeted) mice were crossed to Rosa26^(FLP1) mice(Jax stock #009086) to remove the LacZ/Neo cassette and generate thefloxed allele MAEA^(fl/fl). Both alleles were then backcrossed ontoC57BL/6 background for at least 5 generations before crossing to thevarious Cre strains for functional studies. Genotyping was done by earclip genomic DNA PCR using primers F1+R1+R2 and F2+R3+R4. Primersequences are as follows: F1: gttcagcctcaggattcagg (SEQ ID NO:1); R1:atgagcaggggacctcaac (SEQ ID NO:2); R2: aactgatggcgagctcaga (SEQ IDNO:3); F2: caccagctcaggcagttaca (SEQ ID NO:4); R3: ccacaacgggttcttctgtt(SEQ ID NO:5); R4: cgggaagaagtgggattacc (SEQ ID NO:6).

Antibodies and flow cytometry. Purified goat anti-MAEA polyclonalantibody (I-20) was purchased from Santa Cruz and used at a 1:100concentration. Conjugated donkey anti-goat IgG secondary antibodies werefrom Thermo Fisher and used at a 1:800 concentration.Fluorochrome-conjugated or biotinylated antibodies against mouse Gr-1(Ly6C/G) (clone RB6-8C5), CD115 (clone AFS98), B220 (clone RA3-6B2),F4/80 (clone BM8), VCAM1 (clone 429), CD11b (clone M1/70), CD45 (clone30-F11), Ter119 (clone TER-119), CD71 (clone R17217) and CD44 (cloneIM7), CD45.1 (clone A20), CD45.2 (clone 104), were from BioLegend oreBiosciences. DAPI-negative singlets were analyzed for all live samplesunless otherwise specified. Stained sample suspensions were acquired onan LSR II (BD) and results were analyzed and visualized by FlowJo (TreeStar). For sorting, samples were processed under sterile conditions andsorted on a BD FACSAria.

Generation of MAEA monoclonal antibody (mAb). BALB/c mice were immunizedwith a KLH-conjugated MAEA peptide that is part of the extracellulardomain (AAQKN IDRET SHVTM VVAEL EKTLS GCPA (SEQ ID NO:7)) and boostedwith the same peptide and recombinant MAEA protein (Novus #NBP2-23208).Hybridomas producing mAbs to human MAEA were generated by standardtechniques from splenocytes fused to Ag8.653 or NSO^(bcl2) myelomacells.¹⁹ Clone 92 (IgG2a) was firstly selected by ELISA screen as itsmAbs recognized MAEA peptide/protein, but not human IgG. Subclone 92.25was further selected and validated by FACS staining of wild type but notMAEA^(Csf1rCre) mouse BM cells due to only one amino acid differencebetween the human and mouse sequence in the antibody target region. mAbswere then concentrated and purified from concentrated hybridomasupernatant by Amicon Ultra-15 100K filters (Millipore) and NAb ProteinA/G Spin kits (Thermo Scientific).

Complete blood count. Mice were bled ˜25 μl into an Eppendorf tubecontaining 2 μl of 0.5 M EDTA (Life Technologies) using heparinizedmicro-hematocrit capillary tubes (Fisherbrand) under isofluraneanesthesia. Blood was diluted 1:20 in PBS and analyzed on an Adviacounter (Siemens).

In vivo clearance of RBCs. Long-term RBC clearance was assayed aspreviously described.²⁰ Mice were given a single i.v. NHS sulfo-biotin(Thermo Scientific) injection (100 mg/kg), and the fraction ofbiotinylated RBCs was determined weekly from 1 of blood. Short-termclearance was assayed by i.v. injection of 2×10⁸ CFSE-labelled wild-typeRBCs and monitored at indicated time points.

Splenectomy. Mice were splenectomized under 100 mg/kg ketamine and 10mg/kg xylazine anesthesia as previously described²¹ and allowed torecover for 4 weeks before experiments.

Bone marrow transplantation. All recipient mice were lethally irradiated(600+600 cGy, at least 3 h apart) in a Shepherd Mark 1 irradiator.RBC-lysed bone marrow nucleated cells (1×10⁶, unless otherwiseindicated) were then injected retro-orbitally under isofluraneanesthesia.

Colony-forming assays. Spleen BFU-E was assayed by plating 5×10⁵RBC-lysed splenocytes in MethoCult™ M3436 (Stem Cell Technologies)according to the manufacturer's instructions and colonies wereenumerated on day 10 of culture.

In vivo treatment. For hemolytic anemia induction, mice were injectedi.p. with 40 mg/kg body weight PHZ (Sigma #114715) on day 0 and 1 of theexperiment. For 5-FU challenge, a single dose (250 mg/kg body weight) offreshly made 5-FU was given i.v. to each mouse under isofluraneanesthesia. Mx1-Cre was induced by three doses of PolyI:C (Invivogen)injections every other day at 5 mg/kg i.p. For antibody treatment,purified MAEA mAb and control IgG2a (BioXcell) were diluted in PBS andinjected i.p. at 100 μg daily for 3 weeks.

Cell culture. In vitro terminal differentiation and enucleation ofsorted polychromatic erythroblasts (EB-III) was done as previouslydescribed.²² Briefly, EB-III were FACS sorted and cultured at <10⁶/ml indifferentiation media composed of IMDM, 10% FBS, 1% BSA, 30 ng/ml Epo(BioLegend), 0.2 mg/ml holo-transferrin (Sigma) and 10 μg/ml insulin(ThermoFisher) for 48 hours. At the end of the culture, cells were FACSanalyzed by Ter119 and H33342 staining. In vitro phagocytosis assayusing bone marrow (BMDMs) or spleen (SPDMs) derived macrophages wasslightly modified from previously described.²³ BMDMs or SPDMs wereisolated by adherence from BM or splenic suspensions in macrophage media(RPMI1640 with 10% FBS, 10 mM HEPES and 10 ng/ml M-CSF) for 7days.^(24,25) On day 7, BMDMs, or SPDMs were harvested by gentlescraping and plated at 1×10⁵/well in 12-well plates for 24 hours in fullmacrophage media. The macrophages were then serum-starved for 2 hoursbefore adding 10×10⁶ CFSE-labeled RBCs or 4×10⁴CD45.1 BM cells astarget. After 2-3 hours co-incubation, non-adherent cells were washedand macrophages were scraped for FACS analysis.

Quantification and statistical analysis. In each experiment, each mousewas analyzed as a biological replicate. Data visualization (shown asmean±s.e.m.) and statistical analysis were performed using GraphpadPrism 7. Unpaired Student's t-test was used to assess statisticalsignificance when comparing two samples unless otherwise indicated.

Results

MAEA is required for adult BM macrophage development and EI nicheformation. To examine MAEA function in adult erythropoiesis, mice weregenerated with a floxed allele of MAEA, which leads to a frame shift andnon-sense mediated decay of MAEA mRNA upon Cre-mediated recombination.Flow cytometry analysis using a MAEA-specific polyclonal antibodyrevealed that in adult BM mononucleated cells (BMNCs), MAEA was highlyexpressed in the macrophages with minimal expression by monocytes,neutrophils or B cells (gated as previously described²⁶). MAEA^(fl/fl)mice were intercrossed with a Csf1r-Cre transgenic line¹⁶ to delete MAEAin the monocytic-macrophage lineage. Csf1r-Cre; MAEA^(fl/fl) animals(henceforth MAEA^(Csf1r-Cre)) were born healthy and fertile, andsurvived into adulthood at expected Mendelian ratios, by contrast to theperinatal lethality reported in MAEA-null mice.¹⁴ EfficientMAEA-depletion on BM macrophages was confirmed by FACS analysis. The BMof MAEA^(Csf1r-Cre) mice exhibited a slight, but significant, reductionin cellularity. Further analysis revealed that their BM macrophagenumbers represented ˜30% of wild-type levels. MAEA^(Csf1r-Cre) bonesalso appeared paler than controls, suggesting a reduced erythroidcontent in the marrow. Indeed, the number of BM erythroblasts wasmarkedly reduced in MAEA^(Csf1r-Cre) BM compared to littermate controls.This may be due to disruption of EI formation because there was a markedreduction in EIs (˜30% of control levels) formed in vivo in theMAEA^(Csf1r-Cre) BM. Profiling of the erythroblast maturation statusrevealed a partial block of differentiation at the polychromatic(EB-III) stage.²⁷ These results support a critical role of MAEA in adultBM erythroblastic island formation and functions.

MAEA is dispensable for RBC enucleation. In contrast to the prior reporton MAEA-null mice,¹⁴ peripheral blood anemia was not observed in youngadult MAEA^(Csf1r-Cre) mice. Although a role for MAEA in enucleation asalso been suggested,¹⁴ blood smears and FACS analyses revealed elevatedCD71⁺ Ter119⁺ reticulocyte counts in MAEA^(Csf1r-Cre) animals but nonucleated RBCs in circulating blood. To investigate further this issue,polychromatic erythroblasts (EB-III) were sorted from BM ofMAEA^(Csf1r-Cre) and control mice and enucleation rates were evaluatedin vitro. Cultured MAEA^(Csf1r-Cre)-derived erythroblasts enucleated atsimilar rate as those of controls. These results suggest that MAEAexpression is dispensable for postnatal RBC enucleation.

MAEA regulates RBC dynamics during stress erythropoiesis. Control andMAEA^(Csf1r-Cre) mice were challenged with hemolytic anemia induced bythe hemoglobin-oxidizing reagent phenylhydrazine (PHZ). There was asignificant impairment of the reticulocytosis but not RBC or hematocritrecovery in MAEA^(Csf1r-Cre) mice. Similarly, reticulocytosis inMAEA^(Csf1r-Cre) mice after a single dose of cytotoxic agent5-fluorouracil (5-FU) was significantly delayed while RBC and hematocritshowed an attenuated decline before a mild but significant delay inrecovery. The attenuated early decline in hematocrit was consistent withpreviously described macrophage-depleted models,²⁰ suggesting that MAEAdepletion caused macrophage defects were contributing to both the RBCproduction and clearance. Indeed, the RBC lifespan was significantlyprolonged in MAEA^(Csf1r-Cre) animals. However, no phagocytosis defectsin MAEA^(Csf1r-Cre) macrophages were detected, suggesting the RBCclearance defect likely reflected the overall reduction of macrophagenumbers.

Differential roles of MAEA in spleen and bone marrow macrophages.Interestingly, even though Csf1r-Cre induced similarly efficient MAEAdeletion in splenic red pulp macrophages (RPMs), their numbers were notsignificantly altered. This suggests differential requirements of MAEAin BM and spleen macrophage development or maintenance. Spleen EBnumbers were not significantly altered, although profiling of theirmaturation revealed a similar partial block at the polychromatic stage.However, compensatory stress erythropoiesis was found in the spleen ofMAEA^(Csf1r-Cre) mice as evidenced by splenomegaly and elevatedburst-forming unit-erythroid (BFU-E) numbers.

To further dissect the requirement of MAEA in BM and spleenerythropoiesis, splenectomy was performed on MAEA^(Csf1r-Cre) andlittermate control mice. Splenectomized MAEA^(Csf1r-Cre) animalsdeveloped anemia over the course of 4 weeks while the control groupmaintained healthy peripheral blood counts, suggesting that in thecontext of MAEA^(Csf1r-Cre) animals, the extra-medullary erythropoiesismay mask the phenotype. The splenectomized control and MAEA^(Csf1r-Cre)mice were challenged with PHZ. A severely impaired recovery response wasobserved in MAEA^(Csf1r-Cre) mice. Blood smear again did not reveal anyenucleation defect in the RBCs from splenectomized MAEA^(Csf1r-Cre) micebefore or after PHZ treatment. These results further confirmed that MAEAis critical for adult BM erythropoiesis but not RBC enucleation.

Dominant function of MAEA over VCAM1 in EI niche formation. VCAM1represents another adhesion molecule implicated in EI.^(8,9,20) Tocompare the role of VCAM1 with MAEA, VCAM1 was deleted using Csf1r-Cre.Interestingly, no defects were observed in BM macrophage or erythroblastnumbers in steady state VCAM1^(Csf1r-Cre) mice compared to the controlanimals, except for a minor trend towards reduced spleen erythroblasts.EB maturation as measured by CD44 expression and cell size alsoindicated a normal differentiation profile. The peripheral RBCcompartment during steady state and after PHZ challenge was also normal,consistent with previous studies.^(28,29) These data indicate that MAEAplays a dominant role in BM EI niche function while VCAM1 is dispensablefor adult erythropoiesis in vivo.

Selective MAEA deletion in macrophages, but not erythroblasts, impairsbone marrow erythropoiesis. Csf1r-Cre broadly recombines inhematopoietic stem and progenitor cells (HSPCs), and thus may notdiscriminate between MAEA function in macrophages or erythroblasts thatdescent from HSPCs. To delete selectively MAEA in macrophages,MAEA^(fl/fl) was crossed with CD169-Cre,¹⁷ which does not target theerythroid lineage. MAEA^(CD169-Cre) mice phenotypically mimicked theMAEA^(Csf1r-Cre) animals, with significant reduction of BM macrophageand erythroblast numbers, but no alterations in BM cellularity orcirculating blood parameters. Further analyses revealed a similar defectin in vivo island formation and a partial block in BM EB maturation atthe EB-III stage. Interestingly, while CD169-Cre also recombinedefficiently in spleen RPMs, no significant change in RPM numbers wasobserved and EB numbers in the spleen of MAEA^(CD169-Cre) mice weresignificantly increased suggesting ongoing extra-medullaryerythropoiesis.

By contrast, when MAEA^(fl/fl) was crossed with Epor-Cre, whichrecombines efficiently and selectively in erythroid progenitors,¹⁸MAEA^(Epor-Cre) animals showed significant reductions in circulating RBCcounts with increased mean corpuscular volume (MCV), but no significantchange in BM macrophage and erythroblast numbers or in vivo erythroblastisland formation. In addition, MAEA^(Epor-Cre) EB maturation showed adistinct profile with accumulation of the mature cells in the BM. Noenucleation defect was observed in blood smear or in vitro EB-IIculture. Analysis of the spleen did not reveal significant difference inmacrophage or EB numbers of MAEA^(Epor-Cre) mice, despite efficientEpor-Cre recombination in spleen erythroid lineage. These resultssuggest that MAEA acts in the macrophage, but not erythroblast, tomediate EI formation in adult BM, consistent with the previous report inan in vitro EI reconstitution setting.¹⁴ The result also furtherindicates that MAEA is dispensable for RBC enucleation but may play acell autonomous function in RBC terminal maturation or egress in adultmice.

To investigate the role of macrophage or erythroblast conditional MAEAdeletion in stress erythropoiesis, the two models were challenged withPHZ-induced anemia. Surprisingly, MAEA^(CD169-Cre) mice showed nosignificant difference in hematocrit recovery or reticulocytosis. Thismay be due to the fact that CD169-Cre recombines at a lower frequency(˜60%) in BM macrophages, and/or Csf1r-Cre model is indeed a combinatorymodel of MAEA-deletion in both the macrophages and erythroblasts.Indeed, BM of MAEA^(CD169-Cre) mice showed constantly milder reductionof macrophage and EB numbers than MAEA^(Csf1r-Cre) mice at steady stateand after PHZ. In contrast, MAEA^(Epor-Cre) mice showed a faster declinein RBC content during the early days and a weaker reticulocyte outputduring the later recovery stages. The reduction of RBCs andreticulocytes but normal BM EB numbers after PHZ in MAEA^(Epor-Cre) micealso further suggests that EB MAEA expression may contribute cellautonomously to RBC terminal maturation or egress, but not in EIformation.

Postnatal MAEA deletion or inhibition uncovers important functions inadult erythropoissis. To evaluate the role of MAEA in postnatalerythropoiesis and the maintenance of the EI niche, MAEA was deletedusing the Mx1-Cre line (MAEA^(Mx1-Cre)) in which Cre-mediatedrecombination is inducible by Poly I:C injections. Radiation chimeraswere generated by transplantation of the BM from MAEA^(MX1-Cre) orlittermate controls into wild-type mice to exclude potentialcomplications from the BM microenvironment. Two months aftertransplantation, Cre recombination were induced by three Poly I:Cinjections. Three weeks after the first Poly I:C injection, BMmacrophage and erythroblast numbers were significantly reduced inMAEA^(Mx1-Cre) animals, indicating that MAEA is requiredcell-autonomously for the maintenance of BM macrophages and the EI nicheduring homeostasis.

To investigate further the role of MAEA in erythropoiesis, a novelmonoclonal antibody was developed by immunization of Balb/c mice with apeptide corresponding to the extracellular domain of human MAEA. Clone92.25 (IgG2a) was isolated, which interacts specifically with bothmurine and human MAEA, owing to the highly conserved MAEA amino acidsequence across species (FIG. 18A). Wild-type mice were treated with92.25 or isotype control (100 μg daily 5 days a week for 3 weeks).Anti-MAEA significantly reduced erythroblast numbers in BM withoutaffecting the total cellularity (FIGS. 29B, 29C), but not in the spleen.The treatment also led to alterations in erythroblast differentiationsimilar to macrophage-selective MAEA knockouts (FIG. 30D) withoutreductions of macrophage numbers (FIG. 29E). Furthermore, in vitro EIreconstitution assay showed that 92.25 significantly inhibited islandformation (FIG. 29F), clearly indicating that the EI phenotype from MAEAdeficiency does not originate solely from a defect of macrophagematuration and direct adhesion mediated via MAEA is required for adultBM erythropoiesis.

DISCUSSION

The critical function of EI niche in erythropoiesis was initiallysuggested based on in vitro data^(6,7) and recently confirmed invivo.^(20,30) However, in vivo studies using macrophage depletion cannotdistinguish between the El-dependent and El-independent functions of themacrophage.^(20,30) Several adhesion mechanisms have been proposed tomediate EI formation, providing an ideal model for investigations ofEl-specific functions.^(7,8,10-12) However, studies thus far have beenlargely based on in vitro EI formation assays or germlinegene-deletions. Here it is shown that MAEA is critical for adult BM EIformation and homeostatic erythropoiesis via multiple mechanisms. Bothconstitutive and induced MAEA deletion result in severe reductions of BMmacrophage numbers, indicating that MAEA is required for BM macrophagehomeostasis. Interestingly, spleen macrophages are not affected by MAEAdeletion, in line with recent reports indicating the independence andheterogeneity of tissue resident macrophages under steady state.³¹⁻³³Yet, RBC clearance is delayed in MAEA^(Csf1r-Cre) mice, suggesting arole of macrophages in the BM or other organs that might be affected byMAEA-deletion in RBC clearance. Additionally, antibody inhibitiondisrupted EI formation in vivo and in vitro confirmed that MAEA alsodirectly mediates the adhesion of erythroblasts to macrophages to formEI.¹³⁻¹⁴ BM EB number and maturation profile is significantly impairedeven when macrophage numbers are not affected (such as after anti-MAEAantibody inhibition), suggesting El-specific functions of the macrophagein supporting EB differentiation. The macrophage and erythroidlineage-selective MAEA deletion provides genetic evidence thatMAEA-mediated adhesion is unlikely the result of a homophilicinteraction.

MAEA appears dispensable for the enucleation of adult erythroblasts. Theenucleation process of end-stage erythroid maturation is thought to becoordinated by the sorting and reassembly of nuclear, cytoplasmic andmembrane contents among the resulting reticulocytes andpyrenocytes.^(22,34,35) Previous studies have suggested that MAEA isassociated with actin filaments, preferentially segregating into theextruding pyrenocytes and is required for EB enucleation.^(14,35,36)However, none of the present genetic deletion models have revealed anyenucleation defect in vivo or in vitro, under steady state or afterstress. One possibility is that the erythroid progenitors in previouslyreported MAEA null embryos are so poorly differentiated due to defectsupstream of the EI that they are not able to reach the enucleationstage.^(14,36) Alternatively, the residual expression of MAEA in thepresent conditional knockout models may be masking the phenotypeobserved in the null embryos. It is also tempting to speculate that theenucleation process of fetal erythrocytes may be different from theiradult counterpart, in parallel with their many other differences, suchas the globin compositions.³⁷⁻³¹

Results provided herein provide genetic evidence that the contributionof MAEA in BM EI is dominant compared to that of VCAM1, which whendeleted using the same Csf1r-Cre, is dispensable for macrophagedevelopment, in vivo EI function and erythroid recovery. AlthoughVCAM1-mediated EI formation has commonly been observed invitro,^(8,39,40) its requirement during in vivo erythropoiesis usinggenetic models has not been described.^(28,29) Studies using antibodyinhibition in whole animals have suggested a contribution of VCAM1 inerythropoiesis,²⁰ although VCAM1 expression and function outside of theEl, e.g., in endothelial cells⁴¹ or the hematopoietic stem andprogenitor cell niche,⁴²⁻⁴⁴ cannot be excluded. Since EI mediateserythropoiesis in health and disease,^(20,30) the present studyindicates that MAEA is a promising therapeutic target for erythropoieticdisorders.

Example 6: MAEA is an E3 Ubiquitin Ligase Promoting Autophagy andSelf-Renewal of Hematopoietic Stem Cells

Macrophage-Erythroblast Attacher (MAEA, also known as EMP) wasoriginally identified as an adhesion molecule required forerythroblastic island formation.⁷ Germline deletion of MAEA led tosevere anemia and perinatal mortality.⁹⁷ Sequence analysis indicatesthat MAEA is a highly conserved RING finger domain-containing E3ubiquitin ligase.^(98,99) MAEA's functions, however, remain obscure. Asshown by results provided herein, MAEA is highly expressed inhematopoietic stem cells (HSCs) where it is required for theirmaintenance by restricting cytokine receptor signaling and promotingautophagy. Constitutive MAEA deletion produces severe defects in HSCrepopulation capacity, B- and T-lymphoid differentiation, and prematuredeath of animals from a myeloproliferative syndrome. Postnatal MAEAdeletion leads to transient HSC expansion followed by their depletion.Mechanistically, Applicants found that the surface expression of severalhematopoietic cytokine receptors (e.g., MPL, FLT3) is stabilized inabsence of MAEA, thereby prolonging their intracellular signaling.Additionally, the autophagy flux in HSCs, but not in maturehematopoietic cells, is markedly impaired. Administration ofautophagy-inducing compounds rescued the functional defects ofMAEA-deficient HSCs. Further, MAEA is upregulated in various cancers andassociated with poor survival of acute myelogenous leukemia (AML), andMLL-AF9-driven AML does not develop in the absence of MAEA. Thesefindings thus identify MAEA as an anti-cancer target and novel E3ubiquitin ligase, regulating autophagy, and guarding HSC maintenance.

A conditional MAEA gene deletion recently revealed that MAEA expressionon macrophages, but not erythroblasts, was required for postnatal EIformation.¹⁰⁰ Of note, MAEA was also expressed at high levels on HSCs,and efficiently deleted using Csf1r-Cre¹⁰¹ (FIGS. 31A, 31B; hereafterreferred to as MAEA^(Csf1r-Cre)). MAEA^(Csf1r-Cre) mice are viable butdie prematurely between 4-8 months of age from a myeloproliferativesyndrome characterized by thrombocytosis, anemia and increasedinfiltration of myeloid cells in the lung and liver (FIGS. 30A, 33A,33B). Examination of the bone marrow (BM) at ˜7 months of age revealed anear absence of B-lymphocytes with increased BM cellularity due to Gr-1⁺cell expansion (FIGS. 33C, 33D). By contrast, young adultMAEA^(Csf1r-Cre) mice did not exhibit anemia or a myeloproliferativesyndrome but had a marked reduction (by ˜75%) of circulating leukocytesdue to severe lymphopenia (FIG. 30D). Analysis of their BM revealed asignificant elevation of HSC numbers (CD150⁺ CD48⁻ LSKs) and myeloidprogenitors in MAEA^(Csf1r-Cre) BM compared to control animals, whereasthe lymphoid progenitors were reduced (FIGS. 30E-30G, 34A-34D). Toobtain insight into whether MAEA was required for lymphoid progenitormaintenance or HSC function, single lymphoid-primed multipotentprogenitors (LMPPs) or HSCs were sorted from control andMAEA^(Csf1r-Cre) BM onto OP9 stromal cells to examine their lymphoiddifferentiation potential. These results indicated that MAEA^(Csf1r-Cre)HSCs, but not the LMPPs, showed reductions in lymphoid differentiation(FIG. 30H). These results suggest that HSCs in MAEA^(Csf1r-Cre) mice areskewed toward the myeloid lineage at the expense of the lymphoidpotential.

To evaluate further MAEA's function in HSCs, their ability tocompetitively repopulate the BM of lethally irradiated recipients wasexamined. Surprisingly, despite a higher frequency of phenotypic HSCs,Applicants observed a marked reduction in long-term repopulation ofperipheral blood across all lineages from MAEA^(Csf1r-Cre) donor cellscompared to MAEA^(fl/fl) and MAEA^(fl/+); Csf1r-Cre⁺ control littermates(FIGS. 301, 34E). This was not due to the MHC haplotype mismatch of theCsf1-Cre transgenic mice as Applicants have verified that these animalswere syngeneic for H-2^(b/b) and MAEA^(fl/+); Csf1r-Cre⁺ engrafted aswell as MAEA^(fl/fl). Analysis of recipient BM at 16 weeks aftertransplantation confirmed the severe reduction in the HSC chimerism fromMAEA^(Csf1r-Cre) donors (FIG. 30J). No reduction of MAEA^(Csf1r-Cre)cell homing to the bone marrow was observed (FIG. 34F), suggesting thatMAEA controls HSC repopulation activity.

To confirm these results, HSC function was evaluated after MAEA deletionusing the conditional Mx1-Cre line and poly I:C administration (FIG.30A). During the time course of 3 weeks after the first poly I:Cinjection, results showed that MAEA^(Mx1-Cre) HSCs initially expanded atday 7 followed by a significant reduction compared to control MAEA^(f/f)mice (FIGS. 31B, 35A) while the total BM cellularity was not altered(FIG. 35B). Cell cycle analysis revealed a dramatic loss of quiescenceof MAEA^(Mx1-Cre) HSCs (FIG. 31C) but not of other cell populationsafter poly I:C induction (FIG. 35C). In addition, MAEA^(Csf1r-Cre) HSCswere also actively cycling in young mice while their numbers weredepleted in older mice (FIGS. 35D, 35E). These results suggest MAEAdeletion depletes HSCs likely by aberrant activation, followed by theirexhaustion.

To ascertain the HSC-intrinsic requirement of MAEA, chimeric mice weregenerated by transplantation of an equal mixture of wild-type (CD45.1)and MAEA^(Mx1-Cre) (CD45.2) BM cells into lethally irradiated wild type(CD45.1) recipients and induced MAEA-deletion after stablereconstitution (FIG. 31D). Analysis of the BM and peripheral blood donorchimerism showed a drastic reduction of BM HSCs and LSKs derived fromMAEA^(Mx1-Cre) cells at 2 weeks and a lower peripheral contribution over8 weeks after poly I:C induction (FIGS. 31E, 35F). Similar results wereobtained in MAEA^(Csf1r-Cre) reciprocal transplantation experiments,which indicated that the phenotype was transplantable and did not dependon the BM microenvironment (FIGS. 35G-35L). Thus, these results clearlyshow an intrinsic role of MAEA in HSC maintenance.

Next, the transcriptome of sorted MAEA^(Csf1r-Cre) and littermatecontrol HSCs was analyzed to gain mechanistic insight on how MAEAregulated HSC function. Gene Set Enrichment Analysis (GSEA) revealed astriking up-regulation of gene sets involved in cell activation orproliferation in MAEA^(Csf1r-Cre) HSCs, such as DNA replication, proteinsynthesis/processing and oxidative phosphorylation, but downregulationof several major cell growth-related pathways, including insulin andmTOR signaling FIGS. 31F, 31G, 36A). Consistent with the defectivelymphoid and engraftment potential, the expression of regulators of HSClymphoid potential and maintenance was reduced (FIGS. 31H, 31I). Thetranscriptional downregulation of cell growth-related pathways wascounterintuitive in activated HSCs. Since the activity of these pathwayscould be critically regulated at post-translational levels, downstreamsignaling molecules were assessed by intracellular phospho-flowcytometry. These results revealed a significant downregulation of totalribosomal protein S6, a downstream target of mTOR, in MAEA^(Csf1r-Cre)HSCs compared to control, and a milder reduction of the phosphorylatedS6 (pS6 Ser235/236). This resulted in a significant increase in thepS6/S6 ratio (FIG. 38B), suggesting hyperactivity of mTORC1 signaling.There were no significant changes in pAkt Ser473 or pErk1/2Thr202/Tyr204 basal levels (FIG. 36C).

Based on these analyses, the functional significance of these enrichedpathways were analyzed by treating poly I:C-induced MAEA^(Mx1-Cre) micewith either a proteasome inhibitor (Carfilzomib, CFZ), an inhibitor ofoxidative stress (N-acetylcysteine, NAC), or a mTOR antagonist(rapamycin). Remarkably, while none of the inhibitors significantlyaltered BM cellularity, rapamycin, but not CFZ or NAC, rescued the HSCnumbers after MAEA-deletion (FIGS. 31J, 36D, 36E). Competitivetransplantation experiments from treated and control mice also confirmedthe rescue of functional HSC activity in rapamycin-treatedMAEA^(Mx1-Cre) mice (FIGS. 31K, 36F). Rapamycin, however, did not rescuemacrophage numbers in BM due to MAEA deletion (FIG. 36G).¹⁰⁰ Theseresults suggest that MAEA regulates mTOR activity in HSCs.

Next, experiments were performed to identify how MAEA could interferewith mTOR/intracellular signaling. Although previous studies havesuggested that MAEA might be expressed in the nucleus and/or associatedwith the actin filaments,^(97,103,104) immunofluorescence analysis ofpermeabilized HSCs detected MAEA expression only at the cell surface inlocalized foci (FIG. 31A), raising the possibility that it might beinvolved at surface signaling centers. Recent phylogenetic andbiochemical analyses have suggested that MAEA might be a RINGdomain-containing subunit of a highly conserved E3 ubiquitin ligasecomplex.^(98,99) To further investigate this possibility, theubiquitination landscape in MAEA-deficient and sufficient HSPCs wasinvestigated using an ubiquitin antibody array. Results provided hereindemonstrate that ubiquitinated targets comprising several cell surfacereceptors were significantly reduced in MAEA^(Csf1r-Cre)lineage-negative BM cells compared to control while three targets(Caspase-8, F-box protein 15, and p21Cdkn1a) were significantlyincreased (FIG. 31B). Ubiquitin modifications of protein substrates maylead to proteasome or lysosome-dependent substrate degradation or maymodulate substrate interactome and subcellular localization.¹⁰⁵ Tofurther investigate the functional impact of this change in receptorubiquitination, experiments were performed to focus on the half-life anddownstream signaling of Mpl, cKit, and Flt3, major receptors regulatinghematopoiesis. To this end, BM cells were treated ex vivo with thetranslation inhibitor cycloheximide and evaluated the turnover ofsurface receptors in MAEA^(Csf1r-Cre) and control HSPCs. According toresults provided herein, the half-lives of Flt3 and Mpl, but not cKit,were significantly prolonged in MAEA^(Csf1r-Cre) HSPCs (FIGS. 31C, 37A).Interestingly, a prolonged persistence of pAkt but not pErk was detectedin HSCPs after cytokine stimulation, despite comparable constitutivelevels (FIG. 37B). These results indicate that MAEA negatively modulatesreceptor tyrosine kinase signaling in HSPCs by restricting receptorhalf-lives.

As mTOR inhibitors rescued MAEA-deficient HSCs, experiments weredesigned to determine if the altered protein ubiquitination may lead todysregulation of the downstream lysosome-dependent degradation pathways(e.g., autophagy). Autophagy (macroautophagy), a highly conservedmechanism to recycle macromolecules and organelles via lysosomaldegradation, was suggested to be critical for HSC quiescence andmaintenance.¹⁰⁶⁻¹⁰⁹ Indeed, mTOR signaling and activated Akt arereported to inhibit autophagy, and rapamycin is a potent inducer ofautophagy.^(110,111) Applicants observed no significant change in theexpression of the core autophagy machinery or pro-autophagy genes inMAEA-deficient and sufficient HSCs (FIGS. 38A, 38B). However,experiments discussed herein directly measured autophagy function sinceits regulation mostly occurs at the posttranslational level. Theseexperiments show that the autophagy flux in MAEA^(Csf1r-Cre) HSCs wassignificantly reduced (FIG. 32D). When cultured under starvationconditions or in the presence of rapamycin, wild-type HSCs exhibitedhigh levels of autophagy flux of endogenous LC3-II (distinguishable fromLC3-I through a permeabilization step) compared to more differentiatedcells (FIG. 38B). Interestingly, lineage-negative cells (enriched inHSPCs), but not lineage-positive cells (enriched in mature hematopoieticcells), from MAEA^(Csf1r-Cre) BM presented significant reductions inLC3-II flux (FIG. 38C). In addition, morphometric analyses usingelectron microscopy imaging revealed that the reduced flux was, for themost part, due to a maturation defect in the autophagic compartment ofMAEA^(Csf1r-Cre) HSCs,^(112,113) as Applicants have found a significantreduction in the percentage of autolysosomes (AUT) relative toautophagosomes (APG; FIGS. 31E, 38D) in these cells. Interestingly,albeit more discrete, MAEA^(Csf1r-Cre) HSCs also had a defect inautophagy induction (APG biogenesis) that can explain why APG did notaccumulate in these cells despite the observed maturation defect (FIG.32F). Reduced autophagy flux may explain the lower survival rate ofMAEA-deficient HSCs upon starvation (FIG. 32G).¹¹⁴ To ascertain whetherthe reduced autophagy flux was responsible for the impaired HSC activityin vivo, mTOR-independent autophagy inducers (lithium¹¹⁵ orverapamil¹¹⁶) were administered to poly I:C-induced MAEA^(Mx1-Cre) andcontrol mice. Results provided herein demonstrate that both autophagyinducers could rescue HSC numbers and their repopulation activity (FIGS.32H, 32I, 38E, 38F). These results thus identify MAEA as a criticalregulator of HSC maintenance by enhancing ubiquitination of cytokinereceptors and promoting autophagy.

Next, the human cancer genome atlas (TCGA) database was analyzed toassess MAEA's function in cancer. These analyses revealed a significantassociation of MAEA amplification mutations in many human cancer types(FIG. 25A), and MAEA up-regulation was strongly correlated with poorprognosis in patients with acute myeloid leukemia (AML) andadenocarcinomas of the ovary and lung (FIGS. 25B-25D). MAEA expressionwas also up-regulated in the murine model of MLL-AF9 AML (FIGS. 26A,26B). While wild-type hematopoietic cells overexpressing MLL-AF9 rapidlydeveloped AML, MAEA-deficient cells failed to transform and progressinto full-blown leukemia, indicating that MAEA expression was requiredfor AML engraftment and progression in vivo (FIGS. 26C-26E).Importantly, treatment of AML-bearing mice with a monoclonal anti-MAEAantibody significantly prolonged their survival (FIG. 26K).

Autophagy has been increasingly recognized to be critical for HSCquiescence and maintenance by controlling mitochondria homeostasis,metabolic and oxidative stress.^(109,117) However, there is littleavailable information on the molecular mechanisms that ensure highlevels of autophagy activity in HSCs relative to their downstreamprogeny.¹⁰⁶ Ubiquitination of cellular proteins fine-tunes theirexpression levels, cellular localization and interaction dynamics,thereby influencing many cellular processes.¹¹⁸ Components of theubiquitin proteasome system, mostly E3 ligases, have been suggested toregulate HSC fate by targeting critical signal transducers andtranscription regulators.¹¹⁹ Ubiquitination also plays important rolesin autophagy regulation in other cell systems by influencing thestability and interaction of the autophagy core machinery and selectivecargo recognition,^(120,121) among others. MAEA thus acts as an E3ubiquitin ligase that guards HSC quiescence by restricting cytokinereceptor stability and signaling while ensuring high autophagy flux inHSCs.

Example 7: Anti-MAEA Antibody Therapy Benefits Jak2^(V617F)-InducedPolycythemia Vera (PV)

In this Example, anti-MAEA monoclonal antibody (92.25) or IgG isotypecontrol were injected at 100 μg daily i.p. into control (Ctrl) andJak2^(V617F/V617F) (R/R) mice for one week before analysis. Criticalblood count (CBC) was then analyzed and demonstrates that 92.25injections lowered the reticulocytes, red blood cell counts (RBC), andhemoglobin levels (HGB) in the peripheral blood of Jak2^(R/R) micewithout affecting the control mice (FIG. 42A). In the bone marrow (BM),erythroblast (EB) numbers were reduced by 92.25 injections in bothcontrol and Jak2^(R/R) mice relative to IgG control groups, whilemacrophage numbers were only reduced in the Jak2^(R/R) mice (FIG. 42B).Moreover, 92.25 injection enhanced the maturation of EBs in the BM ofJak2^(R/R) mice without altering the EB maturation profile in wild type(WT) control mice (FIG. 42C).

This is consistent with data presented in Example 5, demonstrating thatMAEA is only required in BM macrophages for EB island function, but notin the spleen. As discussed in greater detail above in Example 5, VCAM1is not required in the BM, however, it may be playing a role in thespleen. Accordingly, a combination therapy may be beneficial to subjectssuffering from Jak2^(V617F)-induced PV.

Throughout this application various publications are referred to insuperscripts. Full citations for these references may be found at theend of the specification before the claims. The disclosures of thesepublications are hereby incorporated by reference in their entiretiesinto the subject application to more fully describe the art to which thesubject application pertains.

From the foregoing, it will be appreciated that specific embodiments ofthe invention have been described herein for purposes of illustration,but that various modifications may be made without deviating from thescope of the invention. Accordingly, the invention is not limited exceptas by the appended claims.

REFERENCES

-   1 Bessis, M. [Erythroblastic island, functional unity of bone    marrow]. Rev Hematol 13, 8-11 (1958).-   2 Hom, J., Dulmovits, B. M., Mohandas, N. & Blanc, L. The    erythroblastic island as an emerging paradigm in the anemia of    inflammation. Immunol Res 63, 75-89, doi:10.1007/s12026-015-8697-2    (2015).-   3 Jacobsen, R. N., Perkins, A. C. & Levesque, J. P. Macrophages and    regulation of erythropoiesis. Curr Opin Hematol 22, 212-219,    doi:10.1097/MOH.0000000000000131 (2015).-   4 de Back, D. Z., Kostova, E. B., van Kraaij, M., van den    Berg, T. K. & van Bruggen, R. Of macrophages and red blood cells; a    complex love story. Front Physiol 5, 9, doi:10.3389/fphys.2014.00009    (2014).-   5 Klei, T. R., Meinderts, S. M., van den Berg, T. K. & van    Bruggen, R. From the Cradle to the Grave: The Role of Macrophages in    Erythropoiesis and Erythrophagocytosis. Front Immunol 8, 73,    doi:10.3389/fimmu.2017.00073 (2017).-   6 Rhodes, M. M., Kopsombut, P., Bondurant, M. C., Price, J. O. &    Koury, M. J. Adherence to macrophages in erythroblastic islands    enhances erythroblast proliferation and increases erythrocyte    production by a different mechanism than erythropoietin. Blood 111,    1700-1708, doi:10.1182/blood-2007-06-098178 (2008).-   7 Hanspal, M. & Hanspal, J. S. The association of erythroblasts with    macrophages promotes erythroid proliferation and maturation: a 30-kD    heparin-binding protein is involved in this contact. Blood 84,    3494-3504 (1994).-   8 Sadahira, Y., Yoshino, T. & Monobe, Y. Very late activation    antigen 4-vascular cell adhesion molecule 1 interaction is involved    in the formation of erythroblastic islands. J Exp Med 181, 411-415    (1995).-   9 Hamamura, K. et al. A critical role of VLA-4 in erythropoiesis in    vivo. Blood 87, 2513-2517 (1996).-   10 Lee, G. et al. Targeted gene deletion demonstrates that the cell    adhesion molecule ICAM-4 is critical for erythroblastic island    formation. Blood 108, 2064-2071, doi:10.1182/blood-2006-03-006759    (2006).-   11 Fabriek, B. O. et al. The macrophage CD163 surface glycoprotein    is an erythroblast adhesion receptor. Blood 109, 5223-5229,    doi:10.1182/blood-2006-08-036467 (2007).-   12 Liu, X. S. et al. Disruption of palladin leads to defects in    definitive erythropoiesis by interfering with erythroblastic island    formation in mouse fetal liver. Blood 110, 870-876,    doi:10.1182/blood-2007-01-068528 (2007).-   13 Hanspal, M., Smockova, Y. & Uong, Q. Molecular identification and    functional characterization of a novel protein that mediates the    attachment of erythroblasts to macrophages. Blood 92, 2940-2950    (1998).-   14 Soni, S. et al. Absence of erythroblast macrophage protein (Emp)    leads to failure of erythroblast nuclear extrusion. J Biol Chem 281,    20181-20189, doi:10.1074/jbc.M603226200 (2006).-   15 Koni, P. A. et al. Conditional vascular cell adhesion molecule 1    deletion in mice: impaired lymphocyte migration to bone marrow. JExp    Med 193, 741-754 (2001).-   16 Qian, B. Z. et al. CCL2 recruits inflammatory monocytes to    facilitate breast-tumour metastasis. Nature 475, 222-225,    doi:10.1038/nature10138 (2011).-   17 Karasawa, K. et al. Vascular-resident CD169-positive monocytes    and macrophages control neutrophil accumulation in the kidney with    ischemia-reperfusion injury. J Am Soc Nephrol 26, 896-906,    doi:10.1681/ASN.2014020195 (2015).-   18 Heinrich, A. C., Pelanda, R. & Klingmuller, U. A mouse model for    visualization and conditional mutations in the erythroid lineage.    Blood 104, 659-666, doi:10.1182/blood-2003-05-1442 (2004).-   19 Sroubek, J. et al. The use of Bcl-2 over-expression to stabilize    hybridomas specific to the HERG potassium channel. J Immunol Methods    375, 215-222, doi:10.1016/j.jim.2011.10.014 (2012).-   20 Chow, A. et al. CD169(+) macrophages provide a niche promoting    erythropoiesis under homeostasis and stress. Nat Med 19, 429-436,    doi:10.1038/nm.3057 (2013).-   21 Reeves, J. P., Reeves, P. A. & Chin, L. T. Survival surgery:    removal of the spleen or thymus. Curr Protoc Immunol Chapter 1, Unit    1 10, doi:10.1002/0471142735.im0110s02 (2001).-   22 Ji, P., Yeh, V., Ramirez, T., Murata-Hori, M. & Lodish, H. F.    Histone deacetylase 2 is required for chromatin condensation and    subsequent enucleation of cultured mouse fetal erythroblasts.    Haematologica 95, 2013-2021, doi:10.3324/haematol.2010.029827    (2010).-   23 Majeti, R. et al. CD47 is an adverse prognostic factor and    therapeutic antibody target on human acute myeloid leukemia stem    cells. Cell 138, 286-299, doi:10.1016/j.cell.2009.05.045 (2009).-   24 Zhang, X., Goncalves, R. & Mosser, D. M. The isolation and    characterization of murine macrophages. Curr Protoc Immunol Chapter    14, Unit 14 11, doi:10.1002/0471142735.im1401s83 (2008).-   25 Davies, J. Q. & Gordon, S. Isolation and culture of murine    macrophages. Methods Mol Biol 290, 91-103 (2005).-   26 Chow, A. et al. Bone marrow CD169⁺ macrophages promote the    retention of hematopoietic stem and progenitor cells in the    mesenchymal stem cell niche. J Exp Med 208, 261-271,    doi:10.1084/jem.20101688 (2011).-   27 Chen, K. et al. Resolving the distinct stages in erythroid    differentiation based on dynamic changes in membrane protein    expression during erythropoiesis. Proc Natl Acad Sci USA 106,    17413-17418, doi:10.1073/pnas.0909296106 (2009).-   28 Ulyanova, T., Phelps, S. R. & Papayannopoulou, T. The macrophage    contribution to stress erythropoiesis: when less is enough. Blood    128, 1756-1765, doi:10.1182/blood-2016-05-714527 (2016).-   29 Ulyanova, T., Jiang, Y., Padilla, S., Nakamoto, B. &    Papayannopoulou, T. Combinatorial and distinct roles of alpha(5) and    alpha(4) integrins in stress erythropoiesis in mice. Blood 117,    975-985, doi:10.1182/blood-2010-05-283218 (2011).-   30 Ramos, P. et al. Macrophages support pathological erythropoiesis    in polycythemia vera and beta-thalassemia. Nat Med 19, 437-445,    doi:10.1038/nm.3126 (2013).-   31 Hashimoto, D. et al. Tissue-resident macrophages self-maintain    locally throughout adult life with minimal contribution from    circulating monocytes. Immunity 38, 792-804,    doi:10.1016/j.immuni.2013.04.004 (2013).-   32 Ginhoux, F. & Guilliams, M. Tissue-Resident Macrophage Ontogeny    and Homeostasis. Immunity 44, 439-449,    doi:10.1016/j.immuni.2016.02.024 (2016).-   33 Hoeffel, G. & Ginhoux, F. Fetal monocytes and the origins of    tissue-resident macrophages. Cell Immunol 330, 5-15,    doi:10.1016/j.cellimm.2018.01.001 (2018).-   34 Konstantinidis, D. G. et al. Signaling and cytoskeletal    requirements in erythroblast enucleation. Blood 119, 6118-6127,    doi:10.1182/blood-2011-09-379263 (2012).-   35 Lee, J. C. et al. Mechanism of protein sorting during    erythroblast enucleation: role of cytoskeletal connectivity. Blood    103, 1912-1919, doi:10.1182/blood-2003-03-0928 (2004).-   36 Soni, S., Bala, S. & Hanspal, M. Requirement for    erythroblast-macrophage protein (Emp) in definitive erythropoiesis.    Blood Cells Mol Dis 41, 141-147, doi:10.1016/j.bcmd.2008.03.008    (2008).-   37 Dame, C. & Juul, S. E. The switch from fetal to adult    erythropoiesis. Clin Perinatol 27, 507-526 (2000).-   38 Dzierzak, E. & Philipsen, S. Erythropoiesis: development and    differentiation. Cold Spring Harb Perspect Med 3, a011601,    doi:10.1101/cshperspect.a011601 (2013).-   39 Yanai, N., Sekine, C., Yagita, H. & Obinata, M. Roles for    integrin very late activation antigen-4 in stroma-dependent    erythropoiesis. Blood 83, 2844-2850 (1994).-   40 Toda, S., Segawa, K. & Nagata, S. MerTK-mediated engulfment of    pyrenocytes by central macrophages in erythroblastic islands. Blood    123, 3963-3971, doi:10.1182/blood-2014-01-547976 (2014).-   41 Yamashita, T. et al. The microenvironment for erythropoiesis is    regulated by HIF-2alpha through VCAM-1 in endothelial cells. Blood    112, 1482-1492, doi:10.1182/blood-2007-11-122648 (2008).-   42 Sturgeon, C. M. et al. Primitive erythropoiesis is regulated by    miR-126 via nonhematopoietic Vcam-1+ cells. Dev Cell 23, 45-57,    doi:10.1016/j.devcel.2012.05.021 (2012).-   43 Dutta, P. et al. Macrophages retain hematopoietic stem cells in    the spleen via VCAM-1. J Exp Med 212, 497-512,    doi:10.1084/jem.20141642 (2015).-   44 Frenette, P. S., Subbarao, S., Mazo, I. B., von Andrian, U. H. &    Wagner, D. D. Endothelial selectins and vascular cell adhesion    molecule-1 promote hematopoietic progenitor homing to bone marrow.    Proc Natl Acad Sci USA 95, 14423-14428 (1998).-   45 Klijn C., et al. A comprehensive transcriptional portrait of    human cancer cell lines. Nat Biotechnol. 2015 March; 33(3):306-12,    Epub 2014 Dec. 8.-   46 Piel F B, Steinberg M H, and Rees D C. Sickle Cell Disease. N    Engl J Med. 2017; 376(16):1561-73.-   47 Ware R E, et al. Sickle cell disease. Lancet. 2017;    390(10091):311-23.-   48 Sundd P, et al. Pathophysiology of Sickle Cell Disease. Annu Rev    Pathol. 2019; 14:263-92.-   49 Zhang D, et al. Neutrophils, platelets, and inflammatory pathways    at the nexus of sickle cell disease pathophysiology. Blood. 2016;    127(7):801-9.-   50 Turhan A, et al. Primary role for adherent leukocytes in sickle    cell vascular occlusion: a new paradigm. Proc Natl Acad Sci USA.    2002; 99(5):3047-51.-   51 Chiang E Y, et al. Imaging receptor microdomains on leukocyte    subsets in live mice. Nat Methods. 2007; 4(3):219-22.-   52 Hidalgo A, et al. Heterotypic interactions enabled by polarized    neutrophil microdomains mediate thromboinflammatory injury. Nat Med.    2009; 15(4):384-91.-   53 Baxter E J, et al. Acquired mutation of the tyrosine kinase JAK2    in human myeloproliferative disorders. Lancet 365, 1054-1061 (2005).-   54 James C, et al. A unique clonal JAK2 mutation leading to    constitutive signaling causes polycythaemia vera. Nature 434,    1144-1148 (2005).-   55 Kralovics R. et al. A gain-of-function mutation of JAK2 in    myeloproliferative disorders. N. Engl. J. Med. 352, 1779-1790    (2005).-   56 Levine R L et al. Activating mutation in the tyrosine kinase JAK2    in polycythemia vera, essential thrombocythemia, and myeloid    metaplasia with myelofibrosis. Cancer Cell 7, 387-397 (2005).-   57 Miyake, K. et al. A VCAM-like adhesion molecule on murine bone    marrow stromal cells mediates binding of lymphocyte precursors in    culture. J Cell Biol 114, 557-565 (1991).-   58 Simmons, P. J. et al. Vascular cell adhesion molecule-1 expressed    by bone marrow stromal cells mediates the binding of hematopoietic    progenitor cells. Blood 80, 388-395 (1992).-   59 Ulyanova, T. et al. VCAM-1 expression in adult hematopoietic and    nonhematopoietic cells is controlled by tissue-inductive signals and    reflects their developmental origin. Blood 106, 86-94 (2005).-   60 Pinho, S. & Frenette, P. S. Haematopoietic stem cell activity and    interactions with the niche. Nat Rev Mol Cell Biol 20, 303-320,    doi:10.1038/s41580-019-0103-9 (2019).-   61 Gurtner, G. C. et al. Targeted disruption of the murine VCAM1    gene: essential role of VCAM-1 in chorioallantoic fusion and    placentation. Genes & development 9, 1-14 (1995).-   62 Frenette, P. S., et al. Endothelial selectins and vascular cell    adhesion molecule-1 promote hematopoietic progenitor homing to bone    marrow. Proceedings of the National Academy of Sciences of the    United States of America 95, 14423-14428 (1998).-   63 Papayannopoulou, T., et al. The VLA4/VCAM-1 adhesion pathway    defines contrasting mechanisms of lodgment of transplanted murine    hemopoietic progenitors between bone marrow and spleen. Proceedings    of the National Academy of Sciences of the United States of America    92, 9647-9651 (1995).-   64 Craddock, C. F., et al. Antibodies to VLA4 integrin mobilize    long-term repopulating cells and augment cytokine-induced    mobilization in primates and mice. Blood 90, 4779-4788 (1997).-   65 Papayannopoulou, T., Priestley, G. V. & Nakamoto, B.    Anti-VLA4/VCAM-1-induced mobilization requires cooperative signaling    through the kit/mkit ligand pathway. Blood 91, 2231-2239 (1998).-   66 Koni, P. A. et al. Conditional vascular cell adhesion molecule 1    deletion in mice: impaired lymphocyte migration to bone marrow. The    Journal of experimental medicine 193, 741-754 (2001).-   67 Deng, L. et al. A novel mouse model of inflammatory bowel disease    links mammalian target of rapamycin-dependent hyperproliferation of    colonic epithelium to inflammation associated tumorigenesis. Am J    Pathol 176, 952-967, doi:10.2353/ajpath.2010.090622 (2010).-   68 Wei, Q. et al. MAEA expressed by macrophages, but not    erythroblasts, maintains postnatal murine bone marrow erythroblastic    islands. Blood 133, 1222-1232, doi:10.1182/blood-2018-11-888180    (2019).-   69 Miyamoto, T. et al. Myeloid or lymphoid promiscuity as a critical    step in hematopoietic lineage commitment. Dev Cell 3, 137-147    (2002).-   70 Sarrazin, S. et al. MafB restricts M-CSF-dependent myeloid    commitment divisions of hematopoietic stem cells. Cell 138, 300-313,    doi:10.1016/j.cell.2009.04.057 (2009).-   71 Dutta, P. et al. Macrophages retain hematopoietic stem cells in    the spleen via VCAM-1. The Journal of experimental medicine 212,    497-512, doi:10.1084/jem.20141642 (2015).-   72 Austin, R., et al. Harnessing the immune system in acute myeloid    leukaemia. Crit Rev Oncol Hematol 103, 62-77,    doi:10.1016/j.critrevonc.2016.04.020 (2016).-   73 Imai, Y et al. Essential roles of VLA-4 in the hematopoietic    system. Int J Hematol 91, 569-575, doi:10.1007/s12185-010-0555-3    (2010).-   74 Kubagawa, H. et al. Biochemical nature and cellular distribution    of the paired immunoglobulin-like receptors, PIR-A and PIR-B. The    Journal of experimental medicine 189, 309-318 (1999).-   75 Takai, T. Paired immunoglobulin-like receptors and their MHC    class I recognition. Immunology 115, 433-440,    doi:10.1111/j.1365-2567.2005.02177.x (2005).-   76 Ujike, A. et al. Impaired dendritic cell maturation and increased    T(H)2 responses in PIR472 B(−/−) mice. Nature immunology 3, 542-548,    doi:10.1038/ni801 (2002).-   77 Ding, Y. B. et al. Association of VCAM-1 overexpression with    oncogenesis, tumor angiogenesis and metastasis of gastric carcinoma.    World J Gastroenterol 9, 1409-1414 (2003).-   78 Lin, K. Y. et al. Ectopic expression of vascular cell adhesion    molecule-1 as a new mechanism for tumor immune evasion. Cancer Res    67, 1832-1841, doi:10.1158/0008-5472.CAN-06-3014 (2007).-   79 Huang, J. et al. Exome sequencing of hepatitis B virus-associated    hepatocellular carcinoma. Nat Genet 44, 1117-1121,    doi:10.1038/ng.2391 (2012).-   80 Yuan, W. et al. Commonly dysregulated genes in murine APL cells.    Blood 109, 961-970, doi:10.1182/blood-2006-07-036640 (2007).-   81 Chen, Q., Zhang, X. H. & Massague, J. Macrophage binding to    receptor VCAM-1 transmits survival signals in breast cancer cells    that invade the lungs. Cancer Cell 20, 538-549,    doi:10.1016/j.ccr.2011.08.025 (2011).-   82 Lu, X. et al. VCAM-1 promotes osteolytic expansion of indolent    bone micrometastasis of breast cancer by engaging    alpha4beta1-positive osteoclast progenitors. Cancer Cell 20,    701-714, doi:10.1016/j.ccr.2011.11.002 (2011).-   83 Damiano, et al. Cell adhesion mediated drug resistance (CAM-DR):    role of integrins and resistance to apoptosis in human myeloma cell    lines. Blood 93, 1658-1667 (1999).-   84 Jacamo, R. et al. Reciprocal leukemia-stroma    VCAM-1/VLA-4-dependent activation of NF-kappaB mediates    chemoresistance. Blood 123, 2691-2702,    doi:10.1182/blood-2013-06-511527 (2014).-   85 Matsunaga, T. et al. Interaction between leukemic-cell VLA-4 and    stromal fibronectin is a decisive factor for minimal residual    disease of acute myelogenous leukemia. Nature medicine 9, 1158-1165,    doi:10.1038/nm909 (2003).-   86 Carlson, P. et al. Targeting the perivascular niche sensitizes    disseminated tumour cells to chemotherapy. Nat Cell Biol 21,    238-250, doi:10.1038/s41556-018-0267-0 (2019).-   87 Krivtsov, A. V. et al. Transformation from committed progenitor    to leukaemia stem cell initiated by MLL-AF9. Nature 442, 818-822,    doi:10.1038/nature04980 (2006).-   88 Jaiswal, S. et al. CD47 is upregulated on circulating    hematopoietic stem cells and leukemia cells to avoid phagocytosis.    Cell 138, 271-285, doi:10.1016/j.cell.2009.05.046 (2009).-   89 Majeti, R. et al. CD47 is an adverse prognostic factor and    therapeutic antibody target on human acute myeloid leukemia stem    cells. Cell 138, 286-299, doi:10.1016/j.cell.2009.05.045 (2009).-   90 Barkal, A. A. et al. Engagement of MHC class I by the inhibitory    receptor LILRB1 suppresses macrophages and is a target of cancer    immunotherapy. Nature immunology 19, 76-84,    doi:10.1038/s41590-017-0004-z (2018).-   91 Barreyro, L. et al. Overexpression of IL-1 receptor accessory    protein in stem and progenitor cells and outcome correlation in AML    and MDS. Blood 120, 1290-1298, doi:10.1182/blood-2012-01-404699    (2012).-   92 Schinke, C. et al. IL8-CXCR2 pathway inhibition as a therapeutic    strategy against MDS and AML stem cells. Blood 125, 3144-3152,    doi:10.1182/blood-2015-01-621631 (2015).-   93 Havel, J. J., Chowell, D. & Chan, T. A. The evolving landscape of    biomarkers for checkpoint inhibitor immunotherapy. Nat Rev Cancer    19, 133-150, doi:10.1038/s41568-019-0116-x (2019).-   94 Woo, S. R., Corrales, L. & Gajewski, T. F. Innate immune    recognition of cancer. Annual review of immunology 33, 445-474,    doi:10.1146/annurev-immunol-032414-112043 (2015).-   95 An, N. & Kang, Y. Using quantitative real-time PCR to determine    donor cell engraftment in a competitive murine bone marrow    transplantation model. J Vis Exp, e50193, doi:10.3791/50193 (2013).-   96 Pinho, S. et al. Lineage-Biased Hematopoietic Stem Cells Are    Regulated by Distinct Niches. Dev Cell 44, 634-641 e634,    doi:10.1016/j.devcel.2018.01.016 (2018).-   97 1 Hanspal, M. & Hanspal, J. S. The association of erythroblasts    with macrophages promotes erythroid proliferation and maturation: a    30-kD heparin-binding protein is involved in this contact. Blood 84,    3494-3504 (1994).-   98 Soni, S. et al. Absence of erythroblast macrophage protein (Emp)    leads to failure of erythroblast nuclear extrusion. J Biol Chem 281,    20181-20189, doi:10.1074/jbc.M603226200 (2006).-   99 Francis, O., Han, F. & Adams, J. C. Molecular phylogeny of a RING    E3 ubiquitin ligase, conserved in eukaryotic cells and dominated by    homologous components, the muskelin/RanBPM/CTLH complex. PLoS One 8,    e75217, doi:10.1371/journal.pone.0075217 (2013).-   100 Lampert, F. et al. The multi-subunit GID/CTLH E3 ubiquitin    ligase promotes cell proliferation and targets the transcription    factor Hbp1 for degradation. Elife 7, doi:10.7554/eLife.35528    (2018).-   101 Wei, Q. et al. MAEA expressed by macrophages, but not    erythroblasts, maintains postnatal murine bone marrow erythroblastic    islands. Blood 133, 1222-1232, doi:10.1182/blood-2018-11-888180    (2019).-   102 Qian, B. Z. et al. CCL2 recruits inflammatory monocytes to    facilitate breast-tumour metastasis. Nature 475, 222-225,    doi:10.1038/nature10138 (2011).-   103 Rossi, D. J. et al. Cell intrinsic alterations underlie    hematopoietic stem cell aging. Proc Natl Acad Sci USA 102,    9194-9199, doi:10.1073/pnas.0503280102 (2005).-   104 Lee, J. C. et al. Mechanism of protein sorting during    erythroblast enucleation: role of cytoskeletal connectivity. Blood    103, 1912-1919, doi:10.1182/blood-2003-03-0928 (2004).-   105 Bala, S. et al. Emp is a component of the nuclear matrix of    mammalian cells and undergoes dynamic rearrangements during cell    division. Biochem Biophys Res Commun 342, 1040-1048,    doi:10.1016/j.bbrc.2006.02.060 (2006).-   106 Kirkin, V., et al. A role for ubiquitin in selective autophagy.    Mol Cell 34, 259-269, doi:10.1016/j.molcel.2009.04.026 (2009).-   107 Warr, M. R. et al. FOXO3A directs a protective autophagy program    in haematopoietic stem cells. Nature 494, 323-327,    doi:10.1038/nature11895 (2013).-   108 Mortensen, M. et al. The autophagy protein Atg7 is essential for    hematopoietic stem cell maintenance. J Exp Med 208, 455-467,    doi:10.1084/jem.20101145 (2011).-   109 Liu, F. et al. FIP200 is required for the cell-autonomous    maintenance of fetal hematopoietic stem cells. Blood 116, 4806-4814,    doi:10.1182/blood-2010-06-288589 (2010).-   110 Riffelmacher, T. & Simon, A. K. Mechanistic roles of autophagy    in hematopoietic differentiation. FEBS J 284, 1008-1020,    doi:10.1111/febs.13962 (2017).-   111 Shimobayashi, M. & Hall, M. N. Making new contacts: the mTOR    network in metabolism and signalling crosstalk. Nat Rev Mol Cell    Biol 15, 155-162, doi:10.1038/nrm3757 (2014).-   112 Chang, Y. Y. et al. Nutrient-dependent regulation of autophagy    through the target of rapamycin pathway. Biochem Soc Trans 37,    232-236, doi:10.1042/BST0370232 (2009).-   113 Reggiori, F. & Ungermann, C. Autophagosome Maturation and    Fusion. J Mol Biol 429, 486-496, doi:10.1016/j.jmb.2017.01.002    (2017).-   114 Eskelinen, E. L. Maturation of autophagic vacuoles in Mammalian    cells. Autophagy 1, 1-10 (2005).-   115 Levine, B. & Kroemer, G. Autophagy in the pathogenesis of    disease. Cell 132, 27-42, doi:10.1016/j.cell.2007.12.018 (2008).-   116 Sarkar, S. et al. Lithium induces autophagy by inhibiting    inositol monophosphatase. J Cell Biol 170, 1101-1111,    doi:10.1083/jcb.200504035 (2005).-   117 Park, H. W. et al. Pharmacological correction of obesity-induced    autophagy arrest using calcium channel blockers. Nat Commun 5, 4834,    doi:10.1038/ncomms5834 (2014).-   118 Ianniciello, A., et al. The Ins and Outs of Autophagy and    Metabolism in Hematopoietic and Leukemic Stem Cells: Food for    Thought. Front Cell Dev Biol 6, 120, doi:10.3389/fcell.2018.00120    (2018).-   119 Lee, M. J. & Yaffe, M. B. Protein Regulation in Signal    Transduction. Cold Spring Harb Perspect Biol 8,    doi:10.1101/cshperspect.a005918 (2016).-   120 Moran-Crusio, K. et al. Regulation of hematopoietic stem cell    fate by the ubiquitin proteasome system. Trends Immunol 33, 357-363,    doi:10.1016/j.it.2012.01.009 (2012).-   121 Shaid, S. et al. Ubiquitination and selective autophagy. Cell    Death Differ 20, 21-30, doi:10.1038/cdd.2012.72 (2013).-   122 Xie, Y. et al. Posttranslational modification of    autophagy-related proteins in macroautophagy. Autophagy 11, 28-45,    doi:10.4161/15548627.2014.984267 (2015).-   123 Eaton et al. Sickle cell hemoglobin polymerization. Adv Protein    Chem, 40: 63-279 (1990).-   124 Steinberg, M H. Management of sickle cell disease. N Engl J Med    340(13): 1021-1030 (1999).-   125 Ballas, S K & Smith, E D. Red blood cell changes during the    evolution of the sickle cell painful crisis. Blood, 79(8) 2154-63    (1992).-   126 Kassim, A A & DeBaun, M R. Sickle cell disease, vasculopathy,    and therapeutics. Annu Rev Med, 64: 451-466 (2013).-   127 Telen et al. Developing new pharmacotherapeutic approaches to    treating sickle-cell disease. Blood 12(1) 239-247 (2006).-   128 Wang et al., Experimental and Therapeutic Medicine (2015).

1. An antibody or immunogenic fragment thereof comprising one or moreof: (a) a heavy chain comprising (SEQ ID NO: 54) SYTMS (CDR1);(SEQ ID NO: 56) EISSGGSYTHYAATVTG (CDR2); and (SEQ ID NO: 58)GELY (CDR3); (b) a heavy chain comprising (SEQ ID NO: 82) SYAMS (CDR1);(SEQ ID NO: 84) EISSTGSYTHYPDTVTG (CDR2); and (SEQ ID NO: 86) GEAL;(c) a light chain comprising (SEQ ID NO: 68) KASQSLLDRGGKTFFN (CDR1);(SEQ ID NO: 70) LVSKLDS (CDR2); and (SEQ ID NO: 72) WQGTHFPWT (CDR3);and (SEQ ID NO: 96) (d) KSSHSLLDSYGKTYLN (CDR1); (SEQ ID NO: 98)LVSKLDS (CDR2); and (SEQ ID NO: 100) WQGTHFPWT (CDR3);

wherein said antibody or immunogenic fragment thereof specifically bindsvascular cell adhesion molecule 1 (VCAM1).
 2. The antibody orimmunogenic fragment thereof of claim 1, comprising one or more of: (a)a VH region comprising the amino acid sequence of SEQ ID NO: 13; (b) aVH region comprising the amino acid sequence of SEQ ID NO: 17; (c) a VLregion comprising the amino acid sequence of SEQ ID NO: 15; and (d) a VLregion comprising the amino acid sequence of SEQ ID NO:
 19. 3. Theantibody or immunogenic fragment thereof of claim 1, comprising:(a) a heavy chain comprising (SEQ ID NO: 54) SYTMS (CDR1);(SEQ ID NO: 56) EISSGGSYTHYAATVTG (CDR2); and (SEQ ID NO: 58)GELY (CDR3); and (b) a light chain comprising (SEQ ID NO: 68)KASQSLLDRGGKTFFN (CDR1); (SEQ ID NO: 70) LVSKLDS (CDR2); and(SEQ ID NO: 72) WQGTHFPWT (CDR3).


4. The antibody or immunogenic fragment thereof of claim 3, comprising aVH region comprising the amino acid sequence of SEQ ID NO:13 and a VLregion comprising the amino acid sequence of SEQ ID NO:15.
 5. Theantibody or immunogenic fragment thereof of claim 1, comprising:(a) a heavy chain comprising (SEQ ID NO: 82) SYAMS (CDR1);(SEQ ID NO: 84) EISSTGSYTHYPDTVTG (CDR2); and (SEQ ID NO: 86) GEAL; and(SEQ ID NO: 96) (b) KSSHSLLDSYGKTYLN (CDR1); (SEQ ID NO: 98)LVSKLDS (CDR2); and (SEQ ID NO: 100) WQGTHFPWT (CDR3).


6. The antibody or immunogenic fragment thereof of claim 5, comprising aVH region comprising the amino acid sequence of SEQ ID NO:17 and a VLregion comprising the amino acid sequence of SEQ ID NO:19.
 7. Theantibody or immunogenic fragment thereof of claim 1, wherein theantibody is selected from the group consisting of a monoclonal antibody,a chimeric antibody, a human antibody, and a humanized antibody.
 8. Afusion protein comprising the antibody or immunogenic fragment thereofof claim
 1. 9. A method of treating sickle cell disease (SCD) in asubject in need thereof comprising administering to the subject theantibody or immunogenic fragment thereof of claim
 1. 10. A nucleic acidcomprising a nucleotide sequence encoding the antibody or immunogenicfragment thereof of claim
 1. 11.-19. (canceled)